Extracts of select endemic plants from the Republic of Mauritius exhibiting anti-cancer and immunomodulatory properties

Mauritius Island possesses unique plant biodiversity with a potential reservoir of biologically active compounds of pharmacological interest. In the current study, we investigated Mauritius endemic plant families Asteraceae, Ebenaceae, Sapotaceae, and Erythroxylaceae, for anti-cancer properties on T cell lymphoma and B16F10 Melanoma cells and immunomodulatory properties on primary T and B cells. The cytotoxicity of methanolic plant extracts at 1, 10, 25 µg/ml was determined. The most active plant species were evaluated for their apoptosis-inducing effects. The immunomodulatory properties of the plants were also studied, and preliminary phytochemical screening of selected plants was done by LC–MS analysis. Psiadia lithospermifolia (Lam.) Cordem (Asteraceae) at 25 µg/ml was the most cytotoxic on both EL4 and B16 cells and triggered apoptosis by the death receptor pathway, and at least in part, by the mitochondrial pathway. Most plant species from Asteraceae, Ebenaceae, Erythroxylaceae, and Sapotaceae inhibited the proliferation of activated T and B cells, although some promoted T cell proliferation. LC–MS profile of Asteraceae plants showed the presence of terpenes, terpenoids, fatty acids, and phenolic. Flavonoids and phenolic acid were also detected from Ebenaceae and Sapotaceae plants. Together, our study demonstrated that Mauritius endemic flora exhibit potential anti-cancer and anti-inflammatory properties worthy of further in-depth studies.

pains, colds, fevers, bronchitis, and asthma 1 . As elaborated in a recent publication 2 , the leaf has the greatest healing properties, commonly used as leaf decoctions or as plaster for immobilizing fractures. Moreover, the Asteraceae species are famously known as a source of sesquiterpene lactones which are reported as promising apoptosis-inducing agents 3 . In Mauritius, the genus Diospyros L is the sole representative of the family Ebenaceae; the plants are used to treat infections caused by bacteria and fungi, against helminthic infections and malaria 4 . The Ebenaceae is commonly known as a prolific source of naphthoquinones such as isodiospyrin, which is a 1, 4-naphthoquinone that is highly cytotoxic isolated from the genus Diospyros 5 . Similarly, the family Erythroxylaceae is represented solely by the genus Erythroxylum P. Browne, whose species are renowned for the treatment of kidney stones 6 . Erythroxylaceae is characterized by the presence of tropane alkaloids such as pervilleines A, which is isolated from Erythroxylum pervillei that has been shown to exhibit promising activity as multidrug resistance (MDR) inhibitor 7 . This study deals with two genera from the Sapotaceae family namely Mimusops L and Sideroxylon L. These plants are used by local people to cure dysentery and diarrhea and to treat skin infections 8 . The triterpenoid saponins are commonly present in Sapotaceae and these compounds have been reported to possess potent cytotoxic activity 9 .
Cancer in Mauritius accounts for 12.8% of deaths recorded in 2016; the most common types of cancer among males is that of the prostate gland (14.2%), lungs (12.6%), and colorectum (12.5%). The main types of cancer among females are that of the breast (40.8%) followed by the uterus (12.7%). Cancer incidence is a big health challenge worldwide and the cancer burden in African countries is projected to rise to over two million cases by 2040. Despite the availability of chemotherapy, successful treatment of cancer remains a big challenge because of the various associated side effects, such as nonselective toxicity against normal cells, and drug resistance. Inflammation is defined as a response to stimulation by infections caused by invading pathogens or endogenous signals such as cell injury and is triggered by innate immunity followed by activation of the adaptive immunity. Inflammatory responses activate T and B lymphocytes resulting in the activation of macrophages, eosinophils, and ultimately secretion of antibodies, activation of complement cascade that plays a critical role in controlling infections and provide a defense to the host. Despite the sophistication of the immune system, when overactivated or not properly terminated, the immune system can cause substantial damage to the host leading to several inflammatory disorders 10 . Moreover, there is now clear evidence to demonstrate that chronic inflammation is also the underlying cause of several types of cancers. Thus, diet and nutrition that suppress chronic inflammation may help prevent the development of such cancers.
In the current study, the anti-cancer and anti-inflammatory properties of several plant extracts from Mauritius were therefore investigated with the goal that such studies would identify compounds that can be used to prevent and treat cancers. The extracts of plant species demonstrating promising tumoricidal activity such as Psiadia lithospermifolia (APL) were selected for further mechanistic study. The current study sheds light on the mode of action of APL through the induction of apoptosis in cancer cells via both the death receptor and mitochondrial pathways in EL4 T cell lymphoma (EL4) and B16F10 (B16) Melanoma cells. The anti-inflammatory and immunomodulatory activity of these extracts on concanavalin A (ConA) activated T cells and LPS-activated B cells from C57BL/6 mice were further investigated. Preliminary LC-MS analysis was also undertaken to identify major phytoconstituents that can explain the therapeutic potential of studied endemic plants.

Material and methods
Collection of plant material. Leaf samples of endemic plant species (Table 1) were collected from different parts of Mauritius Island (Fig. 1B). Voucher specimens were deposited at the Mauritius Herbarium (Ministry of Agro-Industry and Food Security) for accession number/Taxonomic number. The plant's name in Table 1 has been checked on The Plant List website.
Preparation of plant extracts. The collected mature leaf specimens were washed with distilled water and freeze-dried before extraction. Freeze-dried leaves were then ground into a fine powder and stored in air sealed black plastic bags at − 20 °C. The ground powders were extracted using the maceration method. The powder (150 g) was soaked in 450 ml cold methanol in a closed flask with intermittent shaking for 24 h. The mixture www.nature.com/scientificreports/ was filtered, and the residue was further extracted twice by using the same fresh solvent and all the filtrates were pooled together. Finally, from the filtrate, the solvent was evaporated under reduced pressure and low temperature. The resulting dry crude leaf extracts were stored in dark containers at 4 °C. The dry crude leaf extract was further resuspended in DMSO to obtain a working concentration of 1, 10, and 25 µg/ml of dry crude leaf extract.
Mice. C57BL/6 female mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were housed in specific pathogen-free conditions at the AAALAC-accredited animal facility at the University of South Carolina, School of Medicine (Columbia, SC). All animal experiments were carried out in accordance with the National Institutes of Health guidelines and all protocols were approved by the University of South Carolina Institutional Care and Use Committee (IACUC).

Cell lines.
Murine cancer cell lines (EL-4 lymphoma (EL4) and B16F10 Melanoma (B16) cells, were maintained in complete RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 10 mM l-glutamine, 10 mM HEPES, and 100 µg/ml penicillin/streptomycin at 37 °C and 5% CO2, as described in 11,12 . These cell lines had been originally purchased from the American Type Culture Collection (ATCC), Manassas, Virginia. EL-4 cells grow as non-adherent cells while B16F10 cells grow as adherent cells in tissue culture flasks. Thus, when confluent, EL-4 cells were harvested directly while the B16F10 cells were harvested with trypsin/ EDTA, washed three times with the RPMI 1640 medium, and then used in the assays as described previously 11,12 . Reagents. RPMI  www.nature.com/scientificreports/ then incubated at 37 °C for 24 h using 5% CO2 and 95% air. The cells were treated with different concentrations of plant extracts including 1, 10, and 25 µg/ml of dry crude leaf extract. The stock solution was diluted in culture medium ensuring that the DMSO concentration did not exceed 0.1%, a dose at which the cell viability is not affected. Control cells were incubated in culture medium with DMSO as vehicle. The plant extracts were tested in triplicate for their effect on cell viability.
MTT assay. We used the MTT assay to check for the viability of cells. In this assay, cells reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan product. After incubation for 24 h, MTT solution was used to replace the medium as per company protocol. The plates were incubated for an additional 4 h after which the MTT reagent was removed and the formazan crystals were dissolved through the addition of 100% DMSO and gentle shaking. Reading was taken from Victor plate reader at 540 nm.
Annexin V/propidium iodide (PI) apoptosis detection in EL4 and B16 cells treated with plant extracts. This assay was performed as described in our previous studies 13 . The cells were labeled as described by the manufacturer's protocol provided by Alexa Fluor488 Annexin V/PI Cell Apoptosis Kit (Thermo Fisher Scientific Inc., Germany). Cells were treated with 1, 10, 25 μg/ml of dry crude leaf extract for 24 h. After treatment, the cells were rinsed twice with PBS at 4 °C. Next, we added 100 μl of annexin binding buffer to the cells suspended at a concentration of 1 × 10 6 cells per ml followed by incubation with 5 μl Annexin-V of conjugated with Alexa488-and 5 μl of Propidium Iodide (PI) for 15 min in the dark at room temperature. Next, the cells were treated with the binding buffer followed by their analysis using flow cytometry.

TUNEL apoptosis detection in EL4 and B16 cells treated with plant extracts.
The TUNEL assay to detect apoptosis was performed as described previously 12,13 . Briefly, freshly cultured tumor cells were treated with vehicle (DMSO) or different concentrations such as 1, 10, 25 µg/ml of dry crude leaf extract for 24 h. Apoptosis in treated cancer cell lines was determined by performing TUNEL assays (FITC-dUTP nick-end labeling) using an in-situ Cell-Death Detection kit (Roche, Indianapolis, IN).

Role of caspases in apoptosis induction of EL4 and B16 cells. To investigate the role of caspases in
APL-induced Apoptosis in EL4 and B16 cells, we performed in vitro assays as described 12 . Both EL4 and B16 cells were incubated with caspases 8 and 9 inhibitors (20 µM) for at least 1 h before APL treatment. The cells were harvested after 24 h of treatment and TUNEL assays were performed to determine apoptosis as described earlier.

Analysis of mitochondrial membrane potential (MMP). Mitochondrial membrane potential (MMP)
in EL4 and B16 cells after vehicle (DMSO) or APL treatment was determined using 3,3′-Dihexyloxacarbocyanine Iodide (DiOC6) dye as described previously 11,12 . At least three independent experiments were performed, and the data shown represent one of the experiments.

H-Thymidine Uptake Assay for detection of Con A-induced proliferation of T cells and LPS-induced activation of B cells.
Spleens were harvested from C57BL/6 mice. The cells (5 × 10 5 ) were stimulated with Con A (2 μg/ml) or with LPS (100 μg/ml) plated in 96 well round bottom plates, along with different concentration of (1, 10, 25 μg/ml dry crude leaf extract or vehicle (DMSO) for 24 h, after which the cells were harvested and 2 μC i (20 µl) of 3 H-Thymidine was added. The radioactivity was recorded using a liquid scintillation counter (MicroBetaTrilux; PerkinElmer Life and Analytical Sciences).     5D).

Cytotoxic effect of Mauritius endemic plant extracts against EL4 and B16 tumor cells. To
Because there was some background apoptosis in vehicle controls, the data were also expressed to indicate the level of apoptosis in extracts after subtracting the apoptosis seen in vehicle controls (Figs. 4E, 5E). In the absence of caspase inhibitors, APL significantly induced apoptosis in EL4 (73.5%) (Fig. 6D) and B16 cells (80%) (Fig. 7D). However, the data demonstrated significant blocking of APL-induced apoptosis in EL4 cells in the presence of caspase-8 inhibitor (35.3%) (Fig. 6H) and to a lesser extent by caspase-9 inhibitor (63.8%) (Fig. 6L). Similarly, in B16 cells, caspase-8 inhibitor was more effective in blocking apoptosis (31.4%) (Fig. 7H) when compared to caspase-9 inhibi-     www.nature.com/scientificreports/ membrane potential (MMP) using DIOC 6 dye (Figs. 8,9). These data suggested that APL caused significant loss in MMP in both EL-4 and B16 cells although this was not dose-dependent. These data suggested that APL was inducing, at least in part, apoptosis through intrinsic pathway.

Effect of plant extracts on Con-A activated splenocytes. To test if the Mauritius plant extracts can
suppress inflammation, we investigated the effect of extracts on the proliferation of T cells. To that end, we activated spleen cells with T cell mitogen, Concanavalin A (Con-A). The results showed that extracts APA (Fig. 10A), APL (Fig. 10B) and APV (Fig. 10C) from Asteraceae, EDC (Fig. 10E) and EDN (Fig. 10G) from Ebenaceae, EEH (Fig. 10J) from Erythroxylaceae as well as SMB (Fig. 10L) from Sapotaceae decreased ConA-induced T cell proliferation at the higher concentrations of 10 and 25 μg/ml of dry crude leaf extract tested. Interestingly, EDB (Fig. 10D) from Ebenaceae and EEL (Fig. 10K) from Erythroxylaceae showed a dose-dependent increase in proliferative response. The extracts SSB (Fig. 10 M), SSC (Fig. 10N), SSP (Fig. 10O) and SSS (Fig. 10P) from

Effect of plant extracts on B cells.
We also determined the effects of Mauritian plant extracts on the proliferation of splenic B cells using a B cell mitogen, LPS (Fig. 11). The data showed that extracts APA (Fig. 11A), APL (Fig. 11B) and APV (Fig. 11C) from the Asteraceae, EDC (Fig. 11E) from Ebenaceae, EEH (Fig. 11J) from Erythroxylaceae and SSB (Fig. 11M), SSC (Fig. 11N), SSP (Fig. 11O) and SSS (Fig. 11P) from Sapotaceae significantly decreased the LPS-induced B cell proliferation at higher concentration of 25 μg/ml of dry crude leaf extract. However, extracts SMB (Fig. 11L) from Sapotaceae and EDL (Fig. 11F) from Ebenaceae showed biphasic response wherein at low doses, they enhanced B cell proliferation. However, extracts EEL (Fig. 11K) showed dose-dependent increase in proliferative response. The extracts of EDB (Fig. 11D), EDN (Fig. 11G) did not inhibit B cell proliferation at 25 µg/ml of dry crude leaf extract.  Tables 2 and 3. Metabolite assignments were made by comparing the detected compounds MS data in positive and negative mode with cited literature. Identified metabolites (Tables 2, 3) were mainly phenols, flavonoids, terpenes and terpenoids, and fatty acids. The elution order of the chromatographic peaks correlated with decreasing polarity, whereby polar compounds were eluted first followed by semi-polar and finally nonpolar compounds. The chromatogram of Psiadia lithospermifolia in positive mode (Fig. 12A) was characterized by one main region eluting at 20.74 to 26.73 min and in negative mode (Fig. 13A), one main region eluting at 21.6 to 24.97 min with peaks showcasing the occurrence of diterpenes, fatty acids, diterpene acid, and diterpene alcohol. However, the chromatogram of Psiadia arguta shown in positive mode Fig. 12B displayed peaks in two regions: the first half regions (19.81 to 23.10 min) showed polar compounds tentatively identified as flavonoids and the second eluted in the region (25.02 to 34.63 min) indicated diterpenes, alcohol and fatty acid. However, in negative mode, peaks eluted in the region (14.64 to 25.27). Figure 13B indicated the presence of phenolics and fatty acids. In positive mode (Fig. 12C), main peaks at 8.07 to 15.18 min, and in negative mode peaks at 5.24 to 15.95 min (Fig. 13C) for Diospyros nodosa from Ebenaceae were detected mainly as polyphenolic, phenolic acid and flavonoids, whereas the peak at 22.05 min (Fig. 13C) min showed a pentacyclic triterpenoid. Both the positive and negative mode of the chromatogram of Mimusops balata (Figs. 12D, 13D) was dominated in polyphenolic compounds (10.68-13.24 min) ( Table 2) and (9.76 to 13.23 min) ( Table 3). The peak at 18.43 min in negative  (Fig. 13E) (11.48 to 22.96) were mostly flavonoids, terpenoids, and fatty acid. We were unable to identify the only peak (22.43) from Erythroxylum laurifolium in positive mode of the chromatogram (Fig. 12F). However, in negative mode (Fig. 13F) polyphenols and fatty acid were detected in the region (13.24 to 24.99).

Discussion
In the current investigation, the anti-cancer and anti-inflammatory properties of selected Mauritius endemic plant families including Asteraceae, Ebenaceae, Erythroxylaceae, and Sapotaceae traditionally used in the Mauritius folklore as medicines were studied. We also performed a preliminary LC-MS screening to detect the presence of major phytoconstituents from these plants. To study the anti-cancer properties, we investigated the effect of plant extracts to mediate apoptosis in EL-4 lymphoma and B16 melanoma cells. Additionally, to study the anti-inflammatory properties, we tested the effect of the plant extracts on the proliferation of T and B cells. In the current study, we found that a significant number of plant extracts exhibited both anti-cancer and anti-inflammatory effects. Interestingly, some extracts were also able to enhance the proliferation of T and B cells thereby suggesting that such extracts exhibit the properties of immunopotentiators. It was evident from our MTT cytotoxic results that extracts of Asteraceae plants (APA, APV, and APL) at higher doses 25 µg/ml of dry crude leaf extract significantly reduced viability of EL4 and B16 cells compared to other plant families as shown in (Figs. 2A-C, 3A-C). APL turned out to be more cytotoxic than other extracts thereby making it the best candidate for further apoptotic study. Other plant species including Ebenaceae (EDC, EDL and EDB), Erythroxylaceae (EEH and EEL), and Sapotaceae (SMB, SSB, SSC and SSS) were also effective at inhibiting the growth of B16 melanoma cells but not EL-4 cells. Although Mauritius endemic plants are commonly used in the management of health conditions on the island, studies on their mechanisms of anticancer and anti-inflammatory efficacy and action are extremely limited. So far, Mauritius endemic plant families Euphorbiaceae (Acalypha integrifolia Willd) and Myrtaceae (Eugenia tinifolia Lam) have been screened for their anti-cancer properties; they were shown to be cytotoxic against human oesophageal squamous cell carcinoma (KYSE-30) at IC50 of 6.42 ± 2.21 and 6.99 ± 0.38 0 µg LW/ml of extracts respectively 14 . In addition, there is only one study which reported that Psiadia arguta endemic to Mauritius Island and Psiadia dentata (Cass.) DC endemic to Reunion Island, display anti-proliferative effects on human colorectal adenocarcinoma (DLD-1) and human lung carcinoma (A549) 15,16 . However, both plant species turned out to be cytotoxic with IC50 of 24 and 43 µg/ml respectively on human normal fibroblasts. Also, the anti-proliferative properties of Psiadia punctulata (DC.) Vatke. originally from East Africa have also been studied. www.nature.com/scientificreports/ In the current study, we confirmed the apoptotic activity of APL through additional tests such as annexin/PI assay. Our data showed that APL induced early and late apoptosis ultimately leading to necrosis in a time and dose-dependent fashion compared to vehicle-treated controls (Figs. 4, 5). These data suggested that the reductions in the number of viable cells mediated by APL result from induction of apoptosis.
We investigated the mechanisms of apoptosis induced by the methanol extract of APL on EL4 and B16 melanoma cell lines by studying both mitochondrial (intrinsic) and death receptor (extrinsic) pathways. Both pathways are caspase-dependent pathways. In extrinsic pathways, stimulation of external factors promotes the interaction between death receptor (Fas) and Fas ligand (FasL) leading to activation of Caspase 8, which in turn activates the apoptosis executioner, Caspase 3 17 . In intrinsic pathways, the mitochondrial outer membrane is permeabilized in response to intracellular stress leading to the loss of Mitochondrial Membrane Potential and opening of the permeability transition pore. Subsequently, cytochrome C is released from the mitochondria to the cytoplasm which triggers a series of events that activated the apoptotic initiator caspase 9 leading to activation of apoptosis executioner, Caspase 3 18 . In the current study, upon blocking Caspase 8 in EL4 cells treated with methanol extracts of APL, we noted significant blocking of apoptosis (Fig. 6H). A similar trend was noted in APL-induced apoptosis in B16 cells (Fig. 7H). However, when EL4 and B16 cells were treated with APL, decreased www.nature.com/scientificreports/ MMP was also noted (Figs. 8,9), explained by the fact that the apoptosis-inducing compounds found in APL may trigger both pathways of apoptosis, similar to resveratrol that we have reported previously 12 . Alternatively, the APL, being a crude extract, may have multiple apoptosis-inducing compounds as revealed by the LC-MS data, each using only one of the pathways and thus, in combination, promote both pathways of apoptosis. Synergistic anticancer effects are highly appraised for efficient induction of cancer cell death in combination with therapy and delayed drug resistance. Many anti-cancer agents such as cyclophosphamide have also been shown to kill rapidly proliferating cells, such as activated immune cells, thereby acting as anti-inflammatory agents 19 . Moreover, some cancers are driven by chronic inflammation, and thus, suppressing inflammation has a beneficial effect in preventing the development of cancers. To that end, in the current study, we also investigated the effect of Mauritius endemic plant extracts on T and B cells that were activated with specific mitogens such as Con-A and LPS, respectively (Figs. 10,11). T and B lymphocytes are the major components of the adaptive immune response. Our results showed that at a high concentration of 25 µg/ml of dry crude leaf extract, most plant species from Asteraceae (APA, APL, and APV), Ebenaceae (EDC, EDL, EDN, EDE, and EDP), and Erythroxylaceae (EEH), and Sapotaceae www.nature.com/scientificreports/ (SSC, SSP, SSS, and SMB) inhibited the proliferation of Con-A stimulated T cells and LPS-activated B cells. It was interesting to note that most of the plant species which displayed apoptotic action against EL4 or B16 melanoma cells also inhibited the proliferation of T and B cells, previous studies showed that methanol extracts of Psiadia arguta (APA) and Psiadia dentata (Asteraceae) mediated inhibitory activity against Raw 264.7 murine macrophages activated by LPS16. We also noted that some plant extracts from Ebenaceae (EDB) and Erythroxylaceae (EEL) enhanced T cell proliferation in a dose-dependent manner while not affecting B cell proliferation. Plant products have been previously shown to mediate immuno-stimulatory properties. For example, extracts from Tinospora crispa (L.) Hook. f. & Thomson, a tree that belongs to the family Menispermaceae was shown to enhance T cell proliferation induced by ConA 20 . Overall, in the current work, we were able to identify several immunomodulatory properties of extracts from plant families Ebenaceae, Sapotaceae, and Erythroxylaceae. To our knowledge, no studies have been carried out previously to investigate the anti-inflammatory and immunomodulatory properties of these plant species, and thus, our studies form the basis for further investigations on identifying and isolating bioactive molecules and studying their in vivo activities. As a preliminary phytoconstituent screening, we detected the presence of major phytochemicals from selected Psiadia plants using LC-MS analysis and it is not surprising that we detected the presence of terpenes and fatty acids as major components in APL (Psiadia lithospermifolia) (Tables 2, 3). The genus Psiadia from the Mascarene Island is well reputed for the naturally occurring essential oils, which are made up mainly of monoterpenes, sesquiterpenes, aliphatics, and other shikimic acid derivatives but little is mentioned on their potential anti-cancer activity 1,8,21,22 . The presence of cafestol a diterpenoid has previously been shown to induce apoptosis in MSTO-211H 23 . The observed occurrence of geranylgeraniol from APL suggests its role in the inhibitory effects on the proliferation of mitogen-activated T and B cells. Indeed, geranylgeraniol effectively suppressed inflammation in Mevalonate kinase deficiency MKD mouse model 24 . Further, we noted that phenolic compounds predominate in APA, EDN, SMB, and SSC (Table 2). Phenolic compounds are highly appraised for their chemotherapy, chemopreventive, anti-inflammation, and immunomodulation properties. It was no surprise to detect the presence of phenolic compounds among the studied endemic plants. Previous studies have reported the polyphenolic richness of the Mauritius endemic flora 4,22 . Thus, the cytotoxic activities of APA, SMB, and SSC may be attributed to the presence of various phenolic compounds detected. We noted the occurrence of O-methylated flavonol, a flavonoid from APA which has been shown to significantly reduce inflammation in rat paw edema 25 . Interestingly, the LC-MS profile of EDN showed the occurrence of bergenin compounds, which have been shown to inhibit inflammation via the modulation of MAPK and NF-kB signaling pathways in a mouse model of LPS-induced mastitis 26 . On the other hand, another study 27 , showed bergenin to induce Th1 immune responses in a murine model of Mycobacterium tuberculosis infection.