Screening of Apiaceae fruits discovered natural resources with considerable biological potential

Abbreviations: TE, Total methanol Extracts; PE, Petroleum Ether fraction; CL, Chloroform fraction; EA, Ethyl Acetate fraction; ME, Methanol residue fraction; BST, Brine Shrimp Test; FRAP, Ferric Reducing Antioxidant Power * Corresponding authors: goodarzi_s@sina.tums.ac.ir doi: 10.29252/jmp.19.76.46 Received 17 September 2019; Received in revised form 22 August 2020; Accepted 25 August 2020 © 2020. Open access. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/) Research Article


Introduction
Cancer is the second global cause of death after cardiovascular diseases. In 2015, 17.5 million cases were reported with cancer which 8.7 million of them have died. Despite the fact that the mortality from cancers was reduced in many countries, it is expected that its incidence will be increased. So, it is estimated by 2030; there will be 26 million new cancer cases and 17 million cancer deaths per year. Ranking of cancer demonstrated breast, lung and other parts of the respiratory system, and colorectal cancers have more incidences in both sexes [1,2].
According to the prevalence and mortality rate of cancer, the importance of the continuing discovery of new anticancer agents is obvious. The potential of natural products and plantderived compounds for cancer prevention and therapy was the reason for increasing attention to them over the previous few years [3]. On the other hand, over 60% of anti-cancer drugs have origins from natural sources or are related to them [4]. A significant portion of currently used antitumor drugs are synthetic or semi-synthetic derivatives of effective constituents elucidated from plants [5].
Apiaceae (Umbelliferae) family contains about 450 genera and 3700 species which can be found worldwide [6]. Iran is an important center of diversification of Apiaceae plants which is represented by 121 genera, 360 species and 122 endemic taxa. The fruits of this family have many culinary and medicinal properties [7,8].
The purpose of present comprehensive study was toxicity investigation of common spices from Apiaceae fruits by brine shrimp lethally test (BST) and MTT assay. In addition, their antioxidant activities and total phenols were determined and compared between different tribes of Apiaceae family to find and introduce active fraction(s) for further studies.

Extraction and fractionation
The fruits were powdered separately and extracted with 80% methanol via maceration at room temperature. Total methanol extracts (TE) were fractionated with petroleum ether (PE), chloroform (CL) and ethyl acetate (EA) and the residue was named methanol (ME) fraction. Total extracts and fractions were kept at the refrigerator prior to the test.

Brine shrimp lethality test (BST)
The toxicity of total extracts and different fractions of fruits of Apiaceae family were determined by brine shrimp test. The eggs of Artemia salina Leach (Shilat Center, Tehran, Iran) were hatched in aerated 35% saltwater under direct light and warmth (28-30 °C eggs transformed to nauplii 48 h later. Total extracts and various fractions of fruits were dissolved in normal saline (1 % (v/v) DMSO was used when necessary) to obtain different concentrations of 1-500 µg/ml in tubes containing 15 nauplii of brine shrimp. The control group contained the vehicle used for dilutions. Dead larvae were counted in each well after 24 h and mortality percentages (p) were determined according to Abbot's formula, p = pi-C/1-C; where pi was the observed mortality rate of each sample and C means the natural larvae mortality of negative control [10]. The lethal concentration (LC50) value of each sample was calculated and reported.

Cell culture and MTT Assay
The colon carcinoma (HT-29), breast adenocarcinoma (MDA-MB-231) and alveolar basal epithelial adenocarcinoma (A549) cell lines were cultured in RPMI 1640 medium and foreskin fibroblast cells (primary culture) was cultured in DMEM medium containing 10 % Fetal bovine serum (FBS) and 1 % penicillinstreptomycin. Cell cultures were maintained at 37 °C in a humidified 5 % CO2 and 95 % air incubator.
Cytotoxicity studies were performed by MTT assay [11]. Growing cells were incubated into 96-well plates at a density of 1×10 4 cells /well. After 24 h, samples with different concentrations (10-200 μg/ml) were added to wells. 48 h later, 20 μl of 5 mg/ml MTT reagent in phosphatebuffered serum (PBS) was added to each well. The plates were incubated at 37° C for 4 h. Then, the medium was replaced by 100 μl pure DMSO to dissolve formazan crystals which were quantified by reading the absorbance at 570 nm on microplate reader (Anthos, Austria). The cell survival was calculated according to the following equation: % Cell viability = Mean absorbance of sample wells/Mean absorbance of control wells × 100 Three independent experiments were performed for each sample. The concentration of samples inducing 50 % growth inhibition (IC50) was obtained from a dose response curve.

Antioxidant power assay
Antioxidant activities of different fractions of Apiaceae fruits were determined by ferric reducing antioxidant power (FRAP) assay [12]. In this method, ferric tripyridyltriazine (Fe (III)-TPTZ) complex was reduced to its blue colored form (Fe (II)-TPTZ) and the absorbance were measured by spectrophotometer. 50 µl of various fractions of fruits with a concentration of 100 µg/ml were added to 1.5 ml of FRAP reagent and incubated at 37 °C. After 10 min, the absorbance of samples was measured at a wavelength of 593 nm. FRAP reagent used as blank and contained 2.5 ml of TPTZ solution (10 mM) in HCl (40 mM), 2.5 ml of FeCl3 (20 mM) and 25 ml of acetate buffer (0.3 M) with pH 3.6. FeSO4. The standard curve was prepared by FeSO4.7 H2O aqueous solution (125-1000 μM) and antioxidant effects of samples were expressed as mM Fe 2+ /100 g of fractions.

Total phenols determination
Total phenols amounts of different fractions of extract from Apiaceae fruits were determined by Folin-Ciocalteu method [13]. By the addition of molybdo tungstophosphoric heteropoly anion (Folin-Ciocalteu) reagent with yellow color to samples, phenol compounds were oxidized and molybdo tungstophophate with blue color was created. The maximum absorption of blue color was achieved in alkaline pH, usually by the addition of NaHCO3 or Na2CO3. 2 ml of Folin-Ciocalteu reagent (1:10 diluted with distilled water) was mixed with 0.2 ml of methanol solutions of Apiaceae fruits fractions (100 µg/ml). After 5 min, 1.5 ml of sodium bicarbonate solution (60 g/L distilled water) was added to the mixture and incubated at room temperature for 90 min. Then the absorbance was measured at 725 nm by spectrophotometer and the experiment was carried out in triplicate. Different concentrations of Gallic acid methanol solution (10-100 mg/ml) were used for the preparation of standard curve and total phenols of samples were reported as gallic acid equivalents (GAE; mg of Gallic acid per g of samples).

Statistical Analysis
The data were average of three samples measurements and reported as Mean ± SD.
Statistical analysis was performed by Graph Pad Prism 5 via One-way ANOVA and post hoc of Tukey, and P < 0.05 were considered as a significant difference.

Brine shrimp lethality test (BST)
Cytotoxic effects of different fractions from Apiaceae fruits were evaluated by BST and MTT assays. Brine shrimp lethality test is a simple, rapid and valid preliminary screening for definition of bioactive cytotoxic chemicals. Mortality ability of all extracts and fractions on Artemia salina were presented in Table 1.
Statistical analysis showed EA fraction of all plants (mean) demonstrated the best mortality rate and had a significant difference with other fractions (P < 0.05) and PE fraction was the second effective fraction (Fig. 1).
Among EA fractions of plants with 10 µg/ml concentration, B. persicum showed the lowest mortality rate with a significant difference with other EA fractions of plants (P < 0.05).
And among PE fractions with 10 µg/ml concentration, D. carota (carrot) is the best and showed significant difference with other PE fractions of plants (P < 0.05) except A. graveolens (dill).   It was considerable greatest cytotoxic fractions against colon carcinoma (HT-29), breast adenocarcinoma (MDA-MB-231) and alveolar basal epithelial adenocarcinoma (A549) cell lines were from Ammineae and Peucedaneae tribes of Apiaceae family.

Antioxidant power assay
Total antioxidant activities of different fractions of Apiaceae fruits were measured based on FeSO4 standard curve (y = 0.001x + 0.049, r 2 = 0.932) and reported in Table 1

Total phenols determination
Total phenols contents of different fractions of Apiaceae fruits were determined according to the standard curve of Gallic acid (y = 0.0076 x, r 2 = 0.999) and reported in Table 1. Some fractions of P. anisum and T. ammi from Ammineae tribe had the highest phenol contents among others. ME and EA were fractions of fruits with higher amounts of phenols except PE fraction of D. carota and P. anisum (after ME) which contained greatest content of phenols. Statistical analysis of total phenols showed no difference between fractions (P<0.05) (Figure 2).

Discussion
The fruits of Apiaceae family are widely used as spices around the world for culinary and medicinal properties [14,15]. One of the important properties of consumption of plants as food is the prevention of chronic diseases including cancers. In the present study, cytotoxicity, antioxidant effects and total phenols of different fractions of twelve fruits from the Apiaceae family were measured and compared in various tribes of Apiaceae family.
The results were demonstrated cytotoxicity of almost all EA fractions and some other fractions of fruits measured by BST method were comparable with well-known cytotoxic alkaloid, berberine hydrochloride with LC50 of 26 µg/ml [16]. In fruits from Ammineae tribe, EA of A. graveolens, PE and EA of B. persicum, EA and ME of P. crispum, EA of P. anisum and EA of T. ammi had significant cytotoxic activities. A previous study about toxicity investigation on A. salina by traditional medicinal plants of Northern Peru showed LC50 of aqueous and ethanol crude extracts of A. graveolens aerial parts as 171 and 25 µg/ml, respectively [17]. There was an opposite report for none lethality activities of different extracts of A. graveolens of Pakistan on brine shrimp [18]. An investigation in Oman demonstrated ethyl acetate and hydro alcoholic extracts of the leaves of P. crispum killed the shrimp larvae with LC50 values equal to 51.95 and 88.15 μg/ml, respectively [19]. Assessment of brine shrimp cytotoxic activity of T. ammi seeds ethanol extract in Sundarbans mangrove forest region revealed LC50 and LC90 as 35.48 and 66.83 µg/ml, respectively [20]. The toxicity of crude extracts of present study was near to mentioned results.
EA of C. sativum from Smyrneae tribe and EA and CL of F. vulgare from Seselineae tribe were effective against A. salina. Aqueous and ethanol extracts of C. sativum aerial parts with LC50 of 22 and 0.015 µg/ml, and F. vulgare with LC50 of >10000 and 2.75 µg/ml exhibited their BST activity [17]. Another study on Sudanese plants demonstrated LC50 of 893.97 and 0.012 µg/ml for F. vulgare aqueous and ethanol extracts, respectively [21]. The data of present work about crude hydroalcoholic extracts of C. sativum and F. vulgare from Iran showed weaker toxicity than alcoholic extracts of Peru and Sudanese plants.
EA, PE and CL of A. graveolens, EA, PE and CL of H. persicum, and PE and EA of F. assa-foetida from Peucedaneae tribe showed potent effects as cytotoxic agents. Cytotoxicity of the aerial part of A. graveolens (dill) crude extract against brine shrimps showed it was toxic with LC50 value of 51.29 μg/ml [22]. A previous report revealed essential oil of H. persicum (LC50 equal to 0.0071 μl/ml) was known as active fraction in BST assay [23]. Our data about toxicity of crude extract of H. persicum was stronger than previous mentioned result.
EA and CL of C. cyminum, and PE and EA of D. carota from Caucalineae tribe were effective via brine shrimp bioassay. There were no previous reports about brine shrimp bioactivities of these plants.
Further cytotoxic investigations of different fractions of Apiaceae fruits were done by MTT assays against three cancer cell lines (HT-29, MDA-MB-231 and A549) and a normal foreskin fibroblast cell line. According to standard of National Cancer Institute (NCI) and Geran protocol, when a crude extract showed an IC50 less than 20 μg/ml, it was highly cytotoxic and active against cancer cell lines, when IC50 equal to 21-200 μg/ml, it is moderately cytotoxic, when IC50 equal to 201-500 μg/ml, it is weakly cytotoxic and when IC50 is upper than 501 μg/ml, it isn't cytotoxic [24]. In details, all fractions showed IC50 higher than 200 µg/ml (weak or non-cytotoxic) except CL of F. assa-foetida on HT-29 with IC50 equal to 144.16 µg/ml; PE and CL of F. assa-foetida on A549 with IC50 equal to 158.03 and 151.94 µg/ml, respectively and CL of A. graveolens (celery) and F. assa-foetida on foreskin fibroblast cell line with IC50 equal to 193.71 and 140.66 µg/ml, respectively.
Based on this criterion, all fractions of fruits demonstrated no significant cytotoxic effects on cancer and normal cell lines. But there were maybe potent cytotoxic compounds in the most cytotoxic fractions of them including P. anisum, F. assa-foetida, H. persicum, A. graveolens (celery), T. ammi and D. carota, or they were maybe active against other cancer cell lines. It was interesting that most of cytotoxic fractions were from Ammineae and Peucedaneae tribes of Apiaceae. There were little reports about cytotoxic evaluation of extracts or fractions of mentioned plants. Antiproliferative effects of A. graveolens seeds extract has been confirmed on Dalton's lymphoma ascites (DLA) and mouse lung fibroblast (L929) cell lines by induction of apoptosis, DNA fragmentation and morphological changes [25]. Another study showed hexane extract of A. graveolens at concentrations of 100 and 200 μg/ml had the best cytotoxic activity on Rhabdomyosarcoma (RD) cell line [26]. In present research, more fractions of Apiaceae fruits were active against breast cancer (MDA-MB-231) cell line including PE of P. anisum and T. ammi from Ammineae tribe with IC50 equal to 147.76 and 195.83 µg/ml, respectively. Previous investigations showed treatment of liver HepG2 cell lines with anise seeds essential oil could exhibited a significant cytotoxicity [27]. In addition, ethanol extract of P. anisum revealed antiproliferative and apoptotic effects toward human prostate cancer cell line (PC-3) with IC50 value of 400 μg/ml [28]. Cytotoxicity of T. ammi essential oil on colon carcinoma cells was confirmed with an IC50 value of 9.6 µg/ml, too [29]. It was shown phytochemicals including thymol, γ-terpinene and p-cymene played an important role in anticancer activity of essential oil and hexane extracts of ajwain [30].
PE of A. graveolens (dill) and CL of H. persicum and F. assa-foetida from Peucedaneae tribe with IC50 equal to 184.36, 177.61 and 155.65 µg/ml, respectively and PE of D. carota from Caucalineae tribe with IC50 equal to 197.00 µg/ml showed cytotoxicity against breast cancer cell line, too. In before experiment, cytotoxicity evaluation of H. persicum aerial parts essential oil on three human cancer cell lines (HeLa, LS180 and Raji) demonstrated no effects with IC50 more than 2 mg/ml [31]. Cytotoxicity of carrot essential oil was reported by IC50 of 35.3 and 46.1 µg/ml on green monkey kidney (VERO) and human pharynx squamous cell carcinoma (FaDu) cell lines, respectively. Carotol, an important constituent of D. carota essential oil exhibited moderate cytotoxicity on both cell lines with no selectivity [32]. In another study, D. carota essential oil induced selective apoptosis in acute myeloid leukemia (AML) cell line via a MAPK-dependent mechanism [33]. It was interesting that most of cytotoxic plants fractions were from Ammineae and Peucedaneae tribes of Apiaceae. previous study showed methanol extract of A. graveolens seeds exhibited better antioxidant effects as Fe +2 chelating, reducing power activities, and the amounts of total phenols were higher in comparison to other extracts [34]. Another research demonstrated P. anisum extract showed the strongest radical scavenging activity among seven Apiaceae fruits from Iran. In addition, ethyl acetate fraction of P. anisum exhibited the highest antioxidant activity and flavonoid content [35]. P. crispum exhibited DPPH free radical-scavenging activity and cupric reducing antioxidant capacity [36]. There were no reports about antioxidant activities of different fractions of mentioned plants.
Total phenol contents of ME of P. anisum, EA of T. ammi and PE of P. anisum from Ammineae tribe were higher than other samples. According to previous results, anethole and thymol were the most abundant components of P. anisum and T. ammi essential oil which have phenol structures [37,38]. It was interesting that all plants with potent antioxidant activities and high phenol contents were from Ammineae tribe of Apiaceae.

Conclusion
The brine shrimp lethally test is an ideal method in the initial biological screening of a broad range of phytochemical compounds including toxic components. Present investigation demonstrated despite potent larvicidal effects of EA fractions from Apiaceae fruits against A. salina, they didn't demonstrate considerable cytotoxicity against cancer and normal cell lines. Statistical analysis also confirmed that EA fraction is the best cytotoxic fraction and there was no significant difference between all fractions from the point of view of total phenols. Almost more cytotoxic fractions belong to fruits from Peucedaneae tribe while all plants with high phenol contents and antioxidant powers were from Ammineae tribe of Apiaceae. Secondary metabolites in active cytotoxic fractions have potential to act as toxic compounds on other cancer cell lines. In addition, there were not seen significant correlations between cytotoxicity, antioxidant and total phenols of different fractions from Apiaceae fruits.