Phytochemical screening, antioxidant activity and in vitro biological evaluation of leave extracts of Hyptis suaveolens (L.) from south of Mexico

Hyptissuaveolens(L.) is a medicinally plant available in the south and southeast region of Mexico, Latin America and the south and west region of Africa. Infusion of this plant is used to treat diarrhea and as a muscle relaxant. In this study the antioxidant and antibacterial activity, and the phenolic compounds content of Hyptissuaveolens leaves was evaluated. The antioxidant activity was performed by DPPH (1,1-diphenyl-2picryl-hydrazyl) method, obtaining an IC50 range of 2.73 3.58mg/mL for the different prepared extracts. The content of phenolic compounds was carried out by Folin-Ciocalteu method, obtaining for the extracts an oscillation of 253.68 336.50mg of GAE/g of extract. The antibacterial activity was evaluated by disc diffusion method against Staphylococcus aureus, Escherechia coli and Salmonella thipy, of the three pathogenic microorganisms evaluated, S. aureus showed a higher sensitivity to treatment with a minimum inhibitory concentration (MIC) of 0.25mg/mL, for E. coli and S. typhi was 0.5mg/mL. The results confirm the great potential of this species as an antioxidant and antibacterial representing the first contribution to the chemical and biological knowledge of H. suaveolens from Mexican mountain range. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.


Introduction
Antioxidants are substances that delay, control or prevent the initiation and propagation of degenerative diseases in the body and reduce oxidative process (Shah et al., 2014). Phenolic compounds from plant extracts had a strong correlation with antioxidant and antimicrobial activities (Pham et al., 2018) and might have health benefiting properties such as protection against oxidative damage to the cell, minimize tissue damage and improve cell survival (Elghandour et al., 2018;Junsathian et al., 2018). Phenolic compounds inhibit the chain reaction of oxidation by acting as hydrogen donors or free radical acceptors and generation of more stable radicals. The inhibition reaction is considered to be in competition with the propagation step of lipid oxidation and yields stable products that will not initiate new free radicals or bring about a rapid oxidation via a chain reaction (Shahidi and Zhong, 2015).
Hyptissuaveolens (L.)Poit (Fig. 1), is an aromatic plant that was widely cultivated in the pre-Hispanic era of Mexico because its seeds are edible, it is currently cataloged as a "pseudocereal." This species is traditionally known as the donkey's herb and has a wide geographical distribution, in most places where it is found, it is a component of natural vegetation (Vergara-Santana et al., 2005).
The aim of this study was to evaluate the antioxidant and antibacterial activities of the leaves extracts from the plant, and determinate the phytochemical properties and total phenols content thereof. Results from this work will enlighten the medical aspects of this herb.

Instruments
UV spectrophotometer was obtained from VeLab Company, Mexico (Model VE-5100UV). The rotary evaporator was from B€ uchi Instruments, Germany (Model R-100). The incubator was purchased from Felisa company, Mexico (Model: FE-131).

Microbial material
Staphylococcus aureus, E. coli and Salmonella thypistrains were provided and cultured by the Microbiology Department of Faculty of Chemical Sciences, Universidad Veracruzana, Mexico.

Plant material
Hyptissuaveolens (L.) was collected from the central region of Veracruz, Mexico (lowland rainforest, latitude 18°45 0 38 00 N, longitude 96°27 0 38 00 O, altitude 27 m) in October 2018. After collection, the specimen was deposited at the herbarium of the Faculty of Biology (Universidad Veracruzana) in Cordoba, Mexico.
2.5. Leave extract preparation 500 g of leaves were dried at 32°C in a closed room for two weeks and crushed in a fine powder. The powder was poured into a 4 L round bottom flask for maceration at room temperature for three days with 2 Lof the corresponding dissolvent (ethanol, ethyl acetate, trichloromethane, hexane and water).

Phytochemical screening
All the extracts were evaluated using qualitative chemical analysis for the identification of phytochemical species according to the literature (Sarker and Nahar, 2012).

Detection of alkaloids
A sample (5 mL) was stirred with dilute sulfuric acid and four drops of Dragendorff was added. An orange-red precipitated is an indication of the presence of alkaloids.

Determination of coumarins
A small sample of 3 mL was treated with three drops of 5% v/v ethanolic solution of p-dimetilaminobenzaldehyde and hydrogen chloride was bubbled through the mixture. The solution turns orange with the presence of coumarins.

Determination of saponins
An extract sample (5 mL) was diluted with 15 mL of destilled water. The resultant mixture was shaken strongly, the apparition of foam indicates the presence of saponins 2.6.4. Determination of phenylpropanoids A mixture of 2 mL of HCl (0.5 N) and 2 mL of sodium nitrite (10% w/v) was treated with 1 mL of extract and 2 mL NaOH (2 mL). Production of violet color means phenylpropanoids are present.

Determination of flavonoids
An aqueous solution of extract was treated with 10% v/v ammonium hydroxide solution. Apparition of yellow fluorescence color means flavonoids are present.

Determination of tannins
A sample of 5 mL was evaporated and the resultant residues were dissolved in 5 mL of distilled water. The aqueous solution was filtered and a small sample (0.5 mL) was treated with few drops of 5% w/v ferric chloride solution. Production of dark blue color indicates the presence of hydrolysable tannins, and the production of dark green color means condensed tannins are present.

Determination of terpenoids
A small sample of the extract was treated with 1 mL of acetic anhydride, 1 mL of trichloromethane and 1 mL of sulfuric acid. Production of violet color indicates the presence of terpenoids.

Determination of total phenols on the extracts
Total phenol determination was carried out in extracts of ethanol, acidified ethanol (with 1% HCl), ethanol-water (50:50 and 70:30) and water. Extracts were obtained by maceration of leaves powder (30 g) with 200 mL of the corresponding dissolvent for 72 h at room temperature in a dark room. An extract sample of 0.05 g was taken and dissolved in 10 mL of methanol. 0.35 mL of the extract solution was taken in a container and added 1.72 mL of 10% FolinÀCiocalteu's reagent, 1.7 mL of deionized water and 3.45 mL of 7.5% NaHCO 3 solution. The mixture was stirred in a Vortex and heated in a water-bath at 40°C for 15 min. Similarly, the blank sample was prepared without the addition of extract samples. Absorbance of the resultant solution was measured at 760 nm, the same procedure was applied to Gallic acid standard solutions for the preparation of calibration curve. Total phenols content was quantified interpolating the absorbance of the sample on the calibration curve. Results were expressed in terms of Gallic acid equivalent (mg of GAE/g of dried extract).

Antioxidant activity
Antioxidant activity of the leaves extracts was measured by using the DPPH method. 1 mL of extract sample (5 mg/mL) was treated with 2 mL of 24 ppm DPPH solution. The mixture was shaken and incubated in a dark place for 20 min at room temperature. The absorbance of the sample was measured at 515 nm against a blank. The calibration curve was prepared following the same procedure without adding extract. Results are expressed in% of reduced DPPH. The IC 50 of the antioxidant activity was calculated from logarithmic regression of results. The percentage of reduced DPPH was calculated according to the reported method (Mishra et al., 2012).

Determination of minimum inhibitory concentration (MIC)
A sample of 1 mL (0.5 mg) of 70:30 ethanol/water extract was diluted with nutrient broth to obtain solutions with different concentrations (0.5, 0.25, 0.175, 0.0875, 0.043, 0.022 mg/mL). The solutions were inoculated with 2 mL of 0.5 McFarland turbidity standards of Staphylococcus aureus, E. coli and Salmonella thypi at 1 £ 10 8 CFU/mL. The resultant solution was incubated for 24 h at 36°C. After incubation, absorbance was measured at 590 nm. MIC was obtained from the lowest absorbance value observed as indicated in the procedure reported (Bhuyan et al., 2017).

Antibacterial activity
The antibacterial activity of 70:30 ethanol/water extract was evaluated by disc diffusion method. A sample of the extract was diluted in ethanol to give a 1 mg/mL solution. The dissolvent employed for dilution was used as a negative control whilst a 20 mg/mL solution of ciprofloxacin was used as a positive control drug. Filter paper disc with 5 mm of diameter were dipped with the extract solution and the controls. The discs were placed on Mueller-Hinton agar plates inoculated with the bacteria strains and incubated for 24 h at 37°C. The antibacterial activity was evaluated according the diameter of the inhibition zone on the bacterial strains.

Phytochemical screening
The results of phytochemical screening (Table 1) indicated that the ethanolic extract contains tannins, phenylpropanoids and coumarins and the water extract contains saponins, tannins and flavonoids. However, the hexane and trichloromethane extracts only contains terpenoids and the ethyl acetate contains alkaloids and coumarins. In base of the results obtained, the next studies are focused on the ethanolic extract due their phytochemical properties.

Antioxidant activity and total phenols content
A calibration curve of different concentrations of Gallic acid was used to calculate the amount of total phenols of each extract. The antioxidant activity and the quantification of total phenols were carried out only in ethanolic and different ethanol/water extracts. The highest concentration of phenols was found in the ethanolic extract with 336.49 mg of GAE/g and the lowest concentration was present on water extract. The maximum antioxidant activity was found on 70:30 ethanol/water extract, reducing DPPH radicals at 80.76 § 1.2%. Table 2, antioxidant activity increases depending of the polarity of the extract. The calculated IC 50 is represented in Table 2.

Antibacterial activity
Due it antioxidant activity, antibacterial activity was measured only on ethanol-water 70:30 extract. The results (Table 3) show a potential activity against S. aureusand S. typhi, however, the extract does not have activity on E. coli.

Minimum inhibitory concentration
The H. suaveolens ethanol-water 70:30extract presented MIC values for E. Coli and S. typhistrains at 0.5 mg/mL, and for S. aureus at 0.25 mg/mL. The Extract is considered strong inhibitor when it MIC greater than 0.25 mg/Ml. Results are resumed in Fig. 2.

Discussion
The most abundant secondary metabolites are phenolic compounds, enhancing the antioxidant activity against free radicals and others reactive oxygen species. Plants are exposed to highly oxidative    environments that induce the production of radicals, for this reason, they have developed an antioxidant system based on the synthesis of phenolic compounds such flavonoids, tannins and other secondary metabolites. Results of the phytochemical screening are similar to reported in the literature (Gavani and Paarakh, 2008) where is indicated a high concentration of steroids, flavonoids, tannins and alkaloids in ethanol extracts. The antioxidant activity of different extracts of H. suaveolenswas evaluated to determinate the efficacy against free radicals. This property is associated with the capability of secondary metabolites to neutralize all the effects related to free radicals and oxygen reactive species in the body. For that, the total amount of phenolic compounds was determinated. The ethanolic extract showed the highest concentration of phenols while water has the lowest value. The best antioxidant activity was present on the ethanol-water 70:30 extract with the lowest IC 50 value (2.73 mg/mL), according to this parameter, low IC 50 values reflect a high activity antioxidant (Feghhi-Najafabadi et al., 2019), indicating that the accurate mixture of dissolvents allowed to obtain huge amount of metabolites with antioxidant activity.
As a result of the antioxidant activity of the extract, it was used to evaluate its antibacterial activity on important strains in food science. The evaluation of the antimicrobial activity was carried out using the disc diffusion method, where 20 mg of the ethanol-water extract 70:30 of H. suaveolens was used against pathogenic microorganisms such as E. coli, S. typhi and S. aureus. The obtained results showed that the highest inhibition was manifested in strains of S.aureus with a inhibition zone of 0.7 § 0.05 cm, followed by S. typhi with an inhibition zone of 0.52 § 0.12. The E. coli showed to be the most resistant to treatment with the smallest inhibition zone of 0.25 § 0.13 cm.
Due the capability of the extract of H. suaveolens against Gram positive and Gram negative bacteria, the minimum inhibitory concentration (MIC) was determined for find the lowest concentration of the 70:30 ethanolÀwater extract of H. suaveolens that is capable of completely inhibit microbial growth in liquid medium. According to the obtained results, ethanolÀwater 70:30 extract has a MIC of 0.5 mg/mL against E. coli and S. typhi, whilst S.aureus presented a MIC of 0.25 mg/mL. This behavior is because of the presence of phenolic compounds with high activity against Gram positive bacteria.
Results obtained in this work indicate that S. aureus is the microorganism that showed greater sensitivity to the treatment with the ethanol-water extract 70:30 of H. suaveolens, developing inhibition zones of greater diameter compared with that of the other two bacterial strains under study. These results are corroborated with studies previously conducted by Mozhiyarasi and Anuradha (2016) Prasanna and Koppula (2012) for extracts obtained from H. suaveolens, where the polar extracts showed the best antimicrobial activity against Gram-positive bacteria. Also, in studies carried out by Tachakittirungrod and Chowwanapoonpohn (2007) is reported that the essential oil of H. suaveolens has a better antibacterial activity against the Gram positive bacteria S. aureus and S. suis, than against the Gram negative bacteria P. aeuroginosa and E. coli. The low activity in Gram negative bacteria is due to the presence of a bacterial cell structure composed of liposaccharides in addition to peptidoglycan, which prevents the lipophilic passage of essential oils (Mann et al., 2000). It is also known that the mechanisms of resistance are related to the activation of synthesis of the cell wall, with hyperproduction of penicillin-binding proteins, thickening of the wall and the imprisonment of drugs by overproduction of the wall components.
On the contrary, Ríos-Tesch (2015), in a study conducted in Venezuela, point out that the essential oil is active against Gram negative bacteria, showing MIC values that ranged between 300 mg/mL and 450 mL/mL. These differences are observed with respect to the antioxidant and antibacterial activity of H. suaveolens, these differences are attributed to the difference in geographical distribution, climate, soil type, time of year and chemical composition, which varies according to the origin of the species.

Conclusion
These results confirm the great potential of H. suaveolens as antioxidant and antibacterial, and could be used in the treatment of diseases related to oxidative stress and for common infectious diseases caused by bacterial species. This research represents the first contribution to the chemical and biological knowledge of H. suaveolens from Mexico.

Funding sources
This work was financial supported by the Universidad Veracruzana. The authors would like to thank to the Faculty of Biology (Universidad Veracruzana) for the technical support.

Supplementary materials
Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2019.10.016.