Introduction

In recent years, several medicinal plants have been a useful sources of novel biologically active compounds. Many pharmaceutical agents have been discovered by screening natural products from plants, many of which have been developed as new lead egents for pharmaceuticals (Sayed 2000). Examples of successful drugs originally isolated from plants include the Vinca alkaloids from Catharanthus roseus, camptothecin from Camptotheca acuminata, podophyllotoxin from Podophyllum peltatum, and paclitaxel from Taxus baccata (Weber 2009).

Cancer, which is defined as the uncontrolled growth of abnormal cells, is a major health problem. Reactive oxygen species (ROS)-induced cellular damage underlies several key steps in the development of the malignant phenotype, including the evasion of apoptosis, angiogenesis, uncontrolled proliferation, tissue invasion, and metastasis. In addition, many cancers develop resistance to prolonged chemotherapy. Therefore, there is a need to develop more effective and safe therapeutics, such as herbal remedies. Importantly, natural products are a relevant source of novel and potent bioactive compounds with minimal side effects in vivo. Severe oxidative stress has also been implicated in many other chronic and degenerative diseases, including aging, osteoporosis, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (Crespo-Ortiz and Wei 2012; Ziech et al. 2012).

Many phytochemical compounds, including carotenoids and flavonoids, exhibit antioxidant properties. The screening of certain phytochemical compounds is simple and rapid, and requires minimal equipment or selective techniques. Natural antioxidants are capable of inhibiting the formation of carcinogens from precursor substances, and thus could exert preventive effects against cancer (Mukhopadhyay 2007). Oxygen radical absorbance capacity (ORAC) is a method of measuring the antioxidant capacity of biological samples in vitro (Kurihara et al. 2004). The ORAC assay involves calculating the mean free radical activity in samples, and combines both the extent and the length of inhibition time of free radical action by antioxidants. It provides important information regarding the antioxidant capacity of various biological samples from pure compounds such as phenolic and flavonoid substances, to complex matrices such as tea, vegetables, fruits, and animal tissues (Cao et al. 1997; Kurihara et al. 2004).

Lumnitzera racemosa Willd. (Combretaceae) is a small tree found in the mangroves or coast of Asia. The wood of L. racemosa is used as a fuel for its calorific value, whereas its leaves are eaten on South Pacific Island during periods of scarcity. In addition, the reddish brown bark contains ~15 × 19 % tannins, although the leaves and wood contain smaller quantities. Fluid obtained from incisions made in the stems have been applied externally for the treatment of herpes and itches (Anjaneyulu et al. 2003). The major chemical constituents of L. racemosa are polyisoprenoid alcohols, flavonoids, fatty acids, triterpenoids, and tannins (Anjaneyulu et al. 2003). Previous studies reported that extracts from this plant exert various pharmacological activities, including antibacterial (D’Souza et al. 2010), antifungal, antihypertensive (Lin et al. 1993), protein tyrosine phosphatase 1B (PTP1B) inhibitory, hepatoprotective, and antioxidant activities (Ravikumar and Gnanadesigan 2011).

As a part of our ongoing search for bioactive agents in the mangroves, the CH2Cl2– and n-BuOH-soluble fractions of a L. racemosa methanolic extract were selected for phytochemical and biological investigations. We herein wish to report the compounds that may be contributed to the biological activities. The isolation and structural elucidation of thirty-six compounds 1–36 (Fig. 1) from the leaves of L. racemosa, as well as the biological evaluation of these isolates were obtained using peroxyl radical-scavenging and reducing assays, and assessing cytotoxicity in HL-60 and Hel-299 cell lines are reported.

Fig. 1
figure 1

The chemical structures of compounds 1–36 from the leaves of L. racemosa

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Materials and methods

Plant material

The samples of L. racemosa Willd. leaves were collected in Xuan Thuy national botanical park, Namdinh, Vietnam, in July 2013 and identified by Dr. Nguyen The Cuong, IEBR. Voucher specimens (No. XT_CB04C) were deposited at the Institute of Marine Biochemistry, VAST, Vietnam.

Compounds

Bioassay-guided fractionation of the methanolic extract of the mangrove L. racemosa leaves, led to the isolation and structural elucidation thirty-six compounds (1–36). Stock solutions of the tested compounds in DMSO were prepared, kept at −20 °C, and diluted to the final concentration in fresh media before each experiment. For not to affect cell growth, the final DMSO concentration did not exceed 0.5 % in all experiments.

Reducing capacity

Forty microliters of different concentrations of each compound dissolved in ethanol were mixed with 160 μL of a mixture containing 0.5 mM CuCl2 and 0.75 mM neocuproine, a Cu(I) ion specific chelator, in 10.0 mM phosphate buffer, pH 7.4. Absorbance was measured using a microplate reader at 454 nm for 1 h. Increased absorbance of the reaction mixture indicated greater reducing power.

Western blot analysis

The HL-60 (3 × 105 cells/mL) cell was treated with the IC50 values of compounds 1 and 14 for 24 and 48 h. After treatment, the cell was harvested and washed two times with cold PBS. The cell was lysed with lysis buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2.0 mM EDTA, 1.0 mM EGTA, 1.0 mM NaVO3, 10.0 mM NaF, 1.0 mM dithiothreitol, 1.0 mM phenylmethylsulfonylfluoride, 25.0 μg/mL aprotinin, 25.0 μg/mL leupeptin, 1 % Nonidet P-40] and kept on ice for 30 min at 4 °C. The lysates were centrifuged at 15,000 rpm at 4 °C for 15 min. The supernatants were stored at −20 °C until use. Protein content was determined by the Bradford assay (Bradford 1976). The same amount of lysates were separated on 8–15 % SDS-PAGE gels and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) by glycine transfer buffer [192 mM glycine, 25.0 mM Tris–HCl (pH 8.8), and 20 % MeOH (v/v)] at 200 mA for 2 h. After blocking with 5 % nonfat dried milk, the membrane was incubated with primary antibody against Bcl-2 (1:500), Bax (1:1000), cleaved PARP (1:1000), cleaved caspase-9 (1:1000), cleaved caspase-3 (1:1000), ERK1/2 (1:1000), phospho-ERK1/2 (1:1000), c-Myc (1:1000), and β-actin (1:5000) antibodies and incubated with a secondary HRP antibody (1:5000; Vector Laboratories, Burlingame, VT, USA) at room temperature. The membrane was exposed on X-ray films (AGFA, Belgium), and protein bands were detected using a WEST-ZOL® plus Western Blot Detection System (iNtRON, Gyeonggi-do, Republic of Korea).

Statistical analysis

Data are presented as the mean ± SD of at least three independent experiments performed in triplicate. Statistically significant differences were determined by one-way ANOVA followed by Dunnett’s multiple test using GraphPad Prism 6 program (GraphPad Software Inc., San Diego, CA, USA).

Results and discussion

Phytochemical analysis

A methanolic extract of L. racemosa leaves was suspended in water, and then sequentially partitioned using n-hexane, CH2Cl2, and n-BuOH. All of the obtained fractions were evaluated in antioxidant and cytotoxic assays in vitro. The methanolic extract, CH2Cl2–, and n-BuOH-soluble fractions exhibited potent cytotoxic activity with IC50 values of 3.48 ± 0.10, 2.33 ± 0.03, and 1.27 ± 0.06 µg/mL, respectively; other fractions were considered inactive (IC50S > 50 µg/mL). Similarly, this extract and fractions exhibited antioxidant activity in both peroxyl radical-scavenging and reducing assays, with Trolox equivalent (TE) values of ranging from 24.94 ± 0.59 to 28.34 ± 0.20 μM (Fig. 2).

Fig. 2
figure 2

Peroxyl radical-scavenging capacity (A) and reducing capacity (B) of the extracts. Data are expressed as the mean ± standard deviation of three individual experiments. Statistical significance is indicated as determined by one-way ANOVA followed by Dunnett’s multiple comparison test (*P < 0.05, **P < 0.01)

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Therefore, the CH2Cl2– and n-BuOH-soluble fractions were selected for further separation using various chromatographic methods, which resulted in the isolation of thirty-six secondary metabolites (1–36, Fig. 1). Their structures were identified as 1,5,6-trihydroxy-3-methoxyxanthone (1) (Terreaux et al. 1995), 5,6-dihydroxy-2,4-dimethoxyxanthone (2) (Terreaux et al. 1995), perforaphenonoside A (3) (Li et al. 2013), acetylannulatophenonoside (4) (Nedialkov and Kitanov 2002), (6S,9R)-6-hydroxy-3-oxo-α-ionol-9-O-β-d-glucopyranoside (5) (Otsuka et al. 1995), (6S,9R)-9-hydroxy-4,7-megastigmadien-3-one-9-O-(β-d-apiofuranosyl-(1 → 6′)-β-d-glucopyranoside (6) (Tommasi et al. 1992), (6S,9R)-vomifoliol-9-O-β-apiofuranosyl-(1′′ → 6′)-O-β-glucopyranoside (7) (Ito et al. 2001), (+)-pinoresinol (8) (Yeo et al. 2004), polystachyol (9) (Sadhu et al. 2006), (+)-lyoniresinol-3α-O-β-d-glucopyranoside (10) (Inoshiri et al. 1987), (–)-lyoniresinol-3α-O-β-d-glucopyranoside (11) (Inoshiri et al. 1987), 1,6-di-O-p-coumaroyl-β-d-glycopyranoside (12), alangilignoside C (13) (Achenbach et al. 1997), polygalatenoside E (14) (Cheng et al. 2006), 3-(4β-d-glucopyranosyloxy-3-methoxy)-phenyl-2E-propenol (15) (Dubeler et al. 1997), lawsoniaside B (16) (Cuong et al. 2010), icariside F2 (17) (Miyse et al. 1988), benzyl-α-l-arabinopyranosyl-(1 → 6)-β-d-glucopyranoside (18) (Lee et al. 2012), adenosine (19) (Song et al. 2008), quercetine (20) (Satake et al. 2007), quercitrin (21) (Xi-Ning et al. 1997), quercetin-4′-O-β-d-glucopyranosyl-3-O-β-d-glucopyranoside (22) (Jaramillo et al. 2011), myricetin-3-arabinoside (23) (Toshimura et al. 2008), linolenic acid (24) (Mats et al. 1999), (2S)-1-O-α-linolenoyl-2-O-[(7Z,10Z,13Z)-hexadeca-7,10,13-trienoyl]-3-O-β-d-galactopyranosyl-sn-glycerol (25) (Nakabayashi et al. 2009), (2S)-1,2-di-O-[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl]-3-O-β-d-galactopyranosyl-sn-glycerol (26) (Nakabayashi et al. 2009), methyl ester galic acid (27) (Didem et al. 2012), ipuranol (28) (Voutquenne et al. 1999), ergosta-4,6,8(14),22-tetraen-3-one (29) (Gan et al. 1998), stigmasterol (30) (Jash et al. 2013), ginsenoside Re (31) (Fujioka et al. 1989), ginsenoside Rg1 (32) (Fujioka et al. 1989), 20(29)lupen-3-ol (33) (Sholichin et al. 1980), hederagenin 3-O-α-L-arabinopyranoside (34) (Kizu and Tomimori 1982), tormentic acid (35) (Jia et al. 1993), and kaji-ichigoside F1 (36) (Jia et al. 1993) based on spectral and chemical evidence, consistent with literature data. Of these pure isolates, compounds 1–13 were reported for the first time from the genus Lumnitzera, as well as the first report of 14–18 from this plant.

Biological activities

Determination of antioxidant activity

Some biological effects of the mangrove L. racemosa are related to its antioxidant properties. To date, there have been no reports of the antioxidative nature of pure compounds from this plant are available in the literature; most of the publications described the antioxidant activities of only extracts (Ravikumar and Gnanadesigan 2011; Vadlapudi and Chandrasekhr 2009). The antioxidant effects of the isolated compounds were analyzed to evaluate their mode of action. The antioxidant capacity of all isolated compounds (1–36) was determined by measuring their peroxyl radical-scavenging and reducing activities (see Materials and methods and Supporting data).

Our data revealed that 10.0 μg/mL methanolic extract, as well as CH2Cl2 and n-BuOH fractions exhibited potent reducing (with values of 11.73 ± 0.25, 7.47 ± 0.28, and 5.38 ± 0.23 μM TE, respectively), and peroxyl radical-scavenging capacities (with values of 24.94 ± 0.59, 28.34 ± 0.20, and 27.09 ± 0.37 μM TE, respectively, Fig. 2). With regard to peroxyl radical-scavenging activity, Fig. 3a shows that the peroxyl radical-scavenging activities of the isolated compounds on peroxyl radicals generated from AAPH were dose-dependent at concentrations of 1.0 and 10.0 μM. Compounds 4, 8–14, 18, and 20–23 exhibited the most potent activities of the isolated compounds, with TE values of 31.4 ± 0.35, 28.79 ± 0.27, 21.63 ± 0.82, 34.08 ± 0.51, 16.96 ± 0.40, 27.95 ± 0.23, 26.90 ± 0.33, 27.42 ± 0.54, 26.13 ± 0.36, 28.58 ± 0.23, 25.93 ± 0.77, and 10.40 ± 0.68 (μM), respectively, at a concentration of 10.0 μM. The activities of these compounds were higher than that of Trolox, the positive control. In addition, compounds 4, 8, 11, 18, 20, and 22 also exerted significant effects against peroxyl radicals at a concentration of 1.0 μM, with values of 8.55 ± 0.17, 8.96 ± 0.37, 7.74 ± 0.37, 8.60 ± 0.41, 5.28 ± 0.29, and 7.13 ± 0.85 μM TE, respectively (Fig. 3A). A comparison of the peroxyl radical-scavenging ability of the compounds 1–36 revealed an interesting preliminary structure–activity relationship (SAR), where the scavenging capacity was dependent on the location and number of hydroxy groups, and the location and species of sugar moieties in their structures.

Fig. 3
figure 3

Peroxyl radical-scavenging capacity (A) and reducing capacity (B) of the isolated compounds. Data are expressed as the mean ± standard deviation of three individual experiments. Statistical significance is indicated as determined by one-way ANOVA followed by Dunnett’s multiple comparison test (*P < 0.05, **P < 0.01)

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We next assessed the reducing capacity of the compounds by measuring the concentration of Cu (I) ions reduced from Cu (II) ions. The ability of the isolated compounds (1–36) to stimulate the reduction of copper ions was investigated to determine whether their peroxyl radical-scavenging activity, which involves donating a hydrogen atom, was related to their reducing capacity, which entails donating electrons. As the results, compounds 8, 9, 18, and 20–23 (10.0 μM) had meaningful reducing capacity, with 36.02 ± 0.30, 34.09 ± 0.46, 12.49 ± 0.30, 10.73 ± 0.41, 32.87 ± 0.31, 17.23 ± 0.17, and 52.12 ± 0.41 μM of generated copper (I) ions, respectively. The remaining compounds showed negligible effects, with <2.0 μM of copper (I) ions at different concentration (Fig. 3B).

These results suggest that the ability of compounds 4, 8–14, and 18–23 to donate hydrogen atoms or electrons to peroxyl radicals and convert them into relatively stable forms may contribute to their peroxyl radical-scavenging capacities. Although compounds 4, 11, 13, and 14 exhibited potent peroxyl radical-scavenging activities, they demonstrate lower capacities for reducing Cu (II) ions to Cu (I) ions compared with compounds 8, 9, 18, and 20–23. This suggests that the hydrogen-donating ability of compounds 4, 11, 13, and 14 is strong, but that their single electron-transferring activity is weak. This may be due to the presence of only one hydroxyl substituent on the benzene ring, which results in unfavorable electron transfer to the Cu (II) ions. These results demonstrate that lignans (8–13) and flavonoids (20–23), which contain the phenolic hydroxyl groups, are the major components responsible for the potent antioxidant activity of L. racemosa leaf extracts.

Determination of cytotoxic activity

To date, the cytotoxicites of the extracts and/or compounds isolated from L. racemosa have not been reported. Therefore, we assayed the cytotoxic effects of the methanolic extract, as well as CH2Cl2–, and n-BuOH-soluble fractions against HL-60 and Hel-299 cell lines using MTT assays (Table 1). Treating these cell lines with the CH2Cl2 and n-BuOH fractions for 72 h yielded IC50 values ranging from 1.27 ± 0.06 to 3.94 ± 0.55 µg/mL. Notably, the n-BuOH fraction was potently cytotoxic on both human cell lines.

Table 1 The cytotoxic effects of the extracts and isolated compounds 1–36 from L. racemosa
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Next, the cytotoxic effects of all isolated compounds (1–36) from this plant were evaluated against HL-60 and Hel-299 cell lines using MTT assays after treatment for 72 h (Table 1). According to the in vitro results, compounds 1 and 14 exhibited the most potent cytotoxic activity on HL-60 cells, with IC50 values of 0.15 ± 0.29 and 0.60 ± 0.16 μM, respectively. For comparison, the positive control mitoxantrone had IC50 value of 6.80 ± 0.09 μM, respectively. While moderate cytotoxicity was observed with both compounds 1 and 14 against Hel-299 cells (IC50 value of 28.41 ± 2.24 μM), with IC50 values of 55.92 ± 3.51 and 54.34 ± 3.17 μM, respectively. Additionally, compounds 2, 4, 9–13, 15–18, 27, 29, 31, 32, and 34 also exhibited significant cytotoxicity in HL-60 cells, with IC50 values ranging from 2.05 ± 1.20 to 9.11 ± 0.53 μM; but moderate or weak effects were observed on Hel-299 cells. Moderate effects were also observed for compounds 3, 5–8, 15, 20–22, 24–26, and 36 (with IC50 ranging from 12.36 ± 0.56 to 30.52 ± 0.92 μM), whereas the effects of compounds 23, 28, 30, 33, and 35 were weak on both human cell lines compared with the positive control (Table 1). This suggested that active constituents could be isolated from individual compounds, which may become useful for the treatment of leukemia.

Determination of apoptosis in HL-60 human cancer cell line

Many previous reports revealed that natural products and compounds exert potential antitumor activity by inducing apoptosis (Lee et al. 2006). One of the more attractive strategies being considered in current cancer therapy is dietary and/or pharmaceutical manipulation to induce the apoptosis death of malignant cells (Tsuda et al. 2004). An accumulating body of evidence suggests that naturally occurring compounds and many chemotherapeutic agents trigger apoptosis in cancer cells (Bold et al. 1997).

To elucidate the cytotoxic mechanism, we investigated whether the inhibitory effects of compounds 1 and 14 on the proliferation of the human leukemia HL-60 cells arose from the induction of apoptosis (see Supporting data). Apoptotic cells exhibit typical characteristics such as membrane blebbing, cell shrinkage, chromatin condensation, and an increased population of sub-G1 hypodiploid cells (Elmore 2007). Therefore, we examined the apoptotic characteristics of cancer cells following treatment with 1 and 14 at IC50 levels for 48 h. Flow cytometry revealed that the percentage of sub-G1 hypodiploid cells exposed to 1 and 14 was increased significantly after 48 h compared with control (Fig. 4A). These results suggest the induction of apoptosis, which was supported further by an increase in the number of apoptotic bodies in treated cells stained with the cell-permeable DNA dye Hoechst 33342 and visualized using fluorescence microscopy (Fig. 4B).

Fig. 4
figure 4

The degree of apoptosis represented as the DNA content measured by flow cytometric analysis (A) and the fluorescent image of nuclei in HL-60 cell by Hoechst 33342 staining (B). The HL-60 cell was treated with the IC50 values of compounds 1 and 14 for 24 and 48 h. DNA-specific fluorescent dye, Hoechst 33342 (culture medium at a final concentration of 10.0 μg/mL) was directly added to media and apoptotic bodies were observed with an inverted fluorescent microscope equipped with an IX-71 Olympus camera and photographed (magnification × 200)

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The Bcl-2 family is divided into two sub-families that either inhibit or promote apoptosis. The pro-apoptotic members of the Bcl-2 family induce apoptosis by stimulating the release of cytochrome c from the mitochondria, which results in the cleavage and activation of caspase-9. Upon activation, caspase-9 initiates a protease cascade leading to the rapid activation of caspase-3, an effector caspase in cells undergoing apoptosis (Cheng et al. 2005; Hanahan and Weinberg 2000; Zimmermann and Green 2001). Therefore, to determine the possible mechanism underlying the induction of apoptosis, we monitored the expression of the apoptosis-related proteins Bcl-2, Bax, cleaved-caspase-9, caspase-3, and PARP in HL-60 cells. When treated with the IC50 values of 1 and 14, the levels of these apoptosis-related proteins were altered. Specifically, Bcl-2 was decreased, and Bax, and cleaved caspase-9, caspase-3, and PARP were increased in a time-dependent manner (Fig. 5). This suggests that compounds 1 and 14 induced apoptosis by modulating the expression of apoptosis-related proteins in human leukemia HL-60 cells.

Fig. 5
figure 5

The effects of compounds 1 and 14 on the levels of Bcl-2, Bax, cleaved caspase-9, caspase-3, cleaved PARP, and on the activation of ERK1/2 MAPK and c-Myc in HL-60 cell for 24 and 48 h

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The MAPK pathway regulates cell survival, cell growth, and apoptosis. Recent studies reported that the oncoprotein c-Myc was stabilized by activation of the ERK1/2 MAPK pathway. In addition, activation of the PI3K/AKT pathway promoted the transcription of c-Myc (Rasola et al. 2010). To determine if the MAPK pathways played a role in the apoptosis induced by 1 and 14, we examined the expression of ERK1/2 MAPK in HL-60 cells. Treatment with 1 and 14 significantly decreased phospho-ERK1/2 levels significantly (Fig. 5). Several studies have reported that activation of the ERK1/2 MAPK pathway contributes to the stabilization of c-Myc (Yeh et al. 2004). Interestingly, decreased c-Myc and phospho-ERK1/2 levels were observed in HL-60 cells treated with 1 or 14 (Fig. 5). These results suggest that these compounds induce apoptosis in part by inhibiting ERK1/2 signaling and downregulating c-Myc in HL-60 cells.

Conclusions

In conclusion, we investigated the in vitro antioxidant and cytotoxic activities of various fractions of the methanolic extract, and isolated compounds from L. racemosa Willd. leaves. The methanolic extract, CH2Cl2– and n-BuOH-soluble fractions exhibited potent antioxidant and cytotoxic activities. Importantly, the current study has addressed the cytotoxic effects of chemical constituents isolated from the mangrove L. racemosa for the first time. This highlights the need for further investigations of the extracts and isolated compounds from this species for potential use as complementary cancer remedies. An additional mechanism of cancer prevention might be the scavenging of free radical oxygen that are associated with cancer cell development. Therefore, future studies should be performed to investigate the in vitro and/or in vivo mechanisms of action of these compounds.