Phytochemical screening, antioxidant activity and toxicity of the orchids Prosthechea cochleata and Prosthechea livida-A preliminary study

Phytochemical composition, antioxidant, and toxic effects of two orchid (Prosthechea cochleata and Prosthechea livida) were examined in their different hexane and dichloromethane/methanol extracts of leaves, pseudobulbs, rhizome and roots. Phytochemical screening reveals the presence of alkaloids, coumarins, flavonoids, saponins, steroids and terpenes except for anthracene derivatives in P. livida and tannins in both species. Results showed exhibited significant toxicity against brine shrimp with an LC50 values ranged from 3 to 54 μg/ml in 24 h. Finally, the results of the antioxidant test in yeast (Saccharomyces cerevisiae) showed that CH2Cl2:MeOH extract of P. cochleata root extract presented the most efficient protective antioxidant percentage (%PA), with 20 μg/mL, displaying a cell viability of 68.58%. In P. livida case, rhizome extract presented the most efficient %PA, with 1000 μg/mL, with a cell viability of 65.82%. The results evidenced the toxicity of Prosthechea cochleata and Prosthechea livida and reveals potential antioxidative of extracts.


INTRODUCTION
The use of medicinal plants to treat several diseases is a usual practice that is increasing around the world. This knowledge has been used for finding bioactive secondary metabolites, contributing to the development of new drugs. Orchids are a large family of plants with around 25,000 species widespread in the world [1,2]. Orchids are mainly used as ornamental plants. Nevertheless, these plants have played an important role in ancient cultures medicine [3,4]. The research on orchids´ metabolites has been carried out with several species, resulting in the discovering of molecules with a widely chemical activity range [3,4,5,6,7,8]. In Mexico, orchids have been mainly used within the industries of regional handmade crafting and gastronomy, and in the celebration of religious rites, as well.
Orchids are also used in other industries such as medicine as a component to manufacture narcotics, and for medical purposes [3,9].
The genus Prosthechea belongs to the Mesoamerican taxa which is composed of around 90 different species. It is proved that 47 of these species are located in Mexico. Epiphytic plants, occasionally terrestrial, the pseudobulbs are comprised of an internode, single or on group, with 1 to 4 terminal and sessile leaves. These plants present an apical inflorescence, with few or numerous flowers, occasionally showy and with a conspicuous spathe [10]. In recent years, the pharmacological research on Prosthechea has increased due to the potential of these orchids mainly related to the antidiabetic activity, making Prosthechea michuacana one of the most studied orchids. Anti-diabetic, hypoglycemic, antioxidant, anti-inflammatory, and healing properties have been reported to different organic extracts (n-hexane, chloroform, and methanol) and organs of the plant (leaves, pseudobulbs, rhizome, and roots) [11][12][13][14][15][16][17]. Furthermore, the hydroalcoholic extract of flowers, leaves, and pseudobulbs of P. karwinskii reduced the glycemic and lipidemic levels in Wistar rats with induced metabolic syndrome, which was attributed to the extract antioxidant capacity [18,19].

Phytochemical screening, antioxidant activity and toxicity of the orchids Prosthechea cochleata and Prosthechea livida-A preliminary study
In the North of Veracruz Mexico, several species of Prosthechea have been reported [20], among them P. cochleata and P. livida that we can find in abundance in the wild and in cultivation by people from the area. In some cases, they fall from the trees due to the heavy colonies or on the other hand they are knocked down for believing that they are parasitic plants. The present study aimed to evaluate the phytochemical compounds of different extracts and organs of Prosthechea cochelata and P. livida, followed by of the analysis of toxicity in brine-shrimp assay and the potential antioxidant activity in an induced H 2 O 2 yeast model oxidative stress. The plants were divided into leaves, pseudobulbs, rhizomes, and roots. The material was properly washed, cut, and dried before pulverization in a grinder mill, and stored in amber jars at room temperature.

Plant Extracts
To perform the phytochemical analysis of each plant, the hydroalcoholic extracts were prepared separately using 30 g of the dried plant in 100 mL ethanol 70 %. Subsequently, they were sonicated during 5 min and subsequently placed into a water bath at 80°C for 5 min. Each extract was filtered in a Whatman N° 2 filter paper and stored in amber vials. For the pharmacological evaluations (In-vivo antioxidant assay and Brine shrimp cytotoxic assay) of nonpolar and polar metabolites, n-hexane and Dichloromethane:Methanol (CH 2 Cl 2 :MeOH) (1:1) crude extracts were prepared using 100 g of a dried powder from parts of both plants and 100 mL of the solvent extraction by successive macerations (3x for 72 h, each), first with n-hexane and then with the halogen-alcohol solvent. After filtration, extracts were concentrated under a vacuum at 40 °C.
Heat it for few minutes in a bath water. After that, add 3 drops of Dragendorff, Mayer and Wagner reagents in each one of the tubes. A precipitate with/or turbidity confirm alkaloid presence.
Anthraquinones: 0.20 g of dry plant was suspended in 5 mL of chloroform by 15 min. Then, the chloroform extract was divided in two tubes. In one of them, add 1 mL of aqueous solution of sodium hydroxide (5%). A pink color in the solution show the presence of anthraquinone. In the second tube. Add a methanolic solution of magnesium acetate (0.5%). Red or yellow color in aqueous phase confirms anthraquinones.
Coumarins: Put 2 mL of hydroalcoholic extract in a tube. Place a filter paper impregnated with sodium hydroxide (10%) in the mouth of the tube. Heat it in a bath water (100°C) for 5 minutes. Take and dry the filter paper. Examined under UV light, yellow fluorescence being indicative of the presence of coumarins.
Flavonoids: In 2 mL of hydroalcoholic extract was added some fragments of metallic magnesium. Add a few drops of a hydrochloric acid (10%). After to 15 min, add 1 mL of amyl alcohol. The Amyl alcohol staining (yellow to red) is positive evidence for flavonoids.
Saponins: 5 mL of hydroalcoholic extract was evaporated in a tube. Was Added 5 mL of hot water. Cold the solution and shake, rest by 20 min. The presence of foam in the tube is indicative for the presence of saponins.
Steroids: 5 mL was dried and dissolved in 3 mL of chloroform. Was added 2 mL of a solution of acetic anhydride with 2 drops of concentrated sulfuric acid. A green coloration (sometimes blue, violet, pink or red) will be observed that turns to black with time, indicating the presence of steroids.
Triterpenes: 5 mL of hydroalcoholic extract was evaporated in a tube. Add 2 ml of concentrated sulfuric acid and heated for about 2 min. A blood red-yellow coloration is positive for steroids.
Tannins: 5 mL of hydroalcoholic extract was evaporated in a tube. Then, the extract was dissolved with water and filter. Was added some drops of an aqueous solution of ferric chloride (10%). Blue colorations indicate the possible presence of hydrolysable tannins. Green colorations indicate the possible presence of condensed tannins.

In vivo Antioxidant Assay
This test was carried out using the yeast model reported by Golla and Bhimathati. [28], and followed the methodology described by Salgado et al. [29] with minor modifications. The yeast was grown in liquid yeast peptone dextrose (YPD) medium at 28°C to reach an exponential growth phase. The cells inoculum was placed in a 24-wells plates with the n-hexane and CH 2 Cl 2 :MeOH extracts at 1000, 500, 250, 100, 50, and 20 µg/mL to a final volume of 1 mL and DMSO concentration at 5%, which were incubated for 1 h. The treated cells with the extracts were stressed by addition of 4mM hydrogen peroxide prepared in phosphate buffered saline (PBS) solution. Cell viability was calculated by counting colony forming units (CFU) with a cell dilution of 1:10,000 seeded in YPD solid medium plates and incubated for 48h. DMSO (5%) with/without H 2 O 2 4mM were assayed as controls for the antioxidant activity. Experiments were conducted in triplicate and reported as mean of cell percentage of survival, which represents the protective antioxidant activity (PA %). The cell viability after treatment with the extract (% CVE) during 1h and then exposed to H 2 O 2 (% CVP) for 2h, following the formula: (100 / % CVE) x % CVP, was used to determine de PA%.

Brine Shrimp Lethality Assay
The toxic activity of the n-hexane and CH 2 Cl 2 :MeOH extracts was checked by the brine shrimp lethality test by Meyer et al. [30]. The n-hexane and CH 2 Cl 2 :MeOH extracts were dissolved in DMSO to final concentration of 5 % in 0.9% saline solution. The extracts concentrations at 1000, 500, 250, 100, 50, 25, 5, and 1 µg/mL were evaluated in 96-well microplate in triplicate to a volume of 100 µL in sea water. The negative check 5% DMSO and the positive control of dichromate potassium dilutions were used. In each well 10 nauplii (100 µL) were added, and the covered plate was incubated at 22-29 °C for 24 h. Plates were examined under a binocular microscope and the number of dead (non-motile) nauplii in each well were counted and recorded.

Statistics Analysis
The antioxidant activity assay data were analyzed by oneway analysis of variance (ANOVA), followed by Tukey test for comparing the control and the assayed groups, using STATISTICA Version 7.0. Statistical significance was assumed at the level of 0.05. The results are expressed as mean ± standard deviation (SD). The mean results of brine shrimp mortality against logarithm of concentration were plotted using the Microsoft Excel computer program, which also presented regression equations. The regression equations were used to calculate the LC 50 value.

RESULTS AND DISCUSSION
The qualitative phytochemical screening of Prosthechea cochleata and Prosthechea livida showed that the hydroalcoholic extracts were positive for alkaloids, coumarins, flavonoids, saponins, steroids, and triterpenes, excepting for anthraquinones in P. livida, and tannins in both extracts ( Table 1). The general phytochemical characterization of a plant belonging to this genera is shown in P. michuacana, where several constituents as: triterpenoids, stilbenes, phenolics, abietanetype diterpenes from the pseudobulbs [12,16,17], and flavonoids in pseudobulbs [15] were found. Despite abovemencionated, in P. karwinski several flavonoids have been isolated in pseudobulbs, flowers and leaves [18]. As we can see, the pseudobulbs and leaves of the orchids present a great diversity of chemical compounds that could be related to their main functions, which contain the accumulation of nutrients, response to environmental stimuli, and also play basic roles in plant growth [31].
The extracts of P. cochleata and P. livida showed toxicity against brine shrimp with an LC 50 values ranged from 3 to 54 µg/ml in 24 h ( Table 2). The toxicity of the extracts was considered by comparison to Meyer et al. [30] and Clarkson et al. [32] toxicity index where LC 50 values of 500-1000, 100-500, and 0-100 µg/mL were considered low, medium, and highly toxic, respectively. The values obtained in this work according to Meyer or Clarkson could be considered with high toxicity. The use of brine shrimp lethality assay to determinate the toxicity of plant extracts has been considered as a preliminary test [33]. This method is simple, reproducible and can be adapted to do in tube or in 96-well microplate. Several compounds, extracts or cytotoxic agents are toxic to brine shrimp due to sensitivity of this organism [34] (Sasidharan et al. 2008). Coe et al. [35] reported that extracts with presence of alkaloids were cytotoxic, causing brine shrimp death at LC 50 concentrations below 2000 µg/mL, and the extracts without alkaloids the toxicity was due to terpenes.  In this study, all CH 2 Cl 2 : MeOH Prosthechea extracts were positive for alkaloids and other compounds resulting in the toxicity of this.
The CH 2 Cl 2 :MeOH extract of Prosthechea species showed significant antioxidant activity in-vivo by increase the survival percentage of yeast cells against H 2 O 2 ( figure 1 and 2). In P. cochleata, root extract presented the most efficient protective antioxidant percentage (%PA), with 20 µg/mL, displaying a cell viability of 68.58%. In P. livida case, rhizome extract presented the most efficient %PA, with 1000 µg/mL, with a cell viability of 65.82%. The antioxidant activity represented by cell viability may be attributed to the presence of flavonoids observed in the phytochemical screening that were identified in roots of P. cochleata and rhizome of P. livida. In the case of Prosthechea michuacana four flavonoids were isolated by Gutierrez-Perez et al. [14].

CONCLUSIONS
Further studies will be necessary in order to elucidate the chemical constituents of the organic extracts of P. cochleata and P. livida, as well as their pharmacological properties of these molecules for a better understanding about the medicinal potential of these plants. In the present research, the results suggest that the extracts of P. cochleata and P. livida can be used as potential antioxidant on human health as hepatoprotective or anticancer drugs. These extracts also have toxic effects, and it will be necessary to develop further studies to prove their efficacy and safety of its components.