Detection of Antioxidant and Antimicrobial Activities in Phenol Components and Essential oils of Cistus ladaniferus and Mentha Suaveolens Extracts

1Laboratory of Natural Resources and Environment, Polydisciplinary Faculty of Taza, Sidi Mohamed Ben Abdellah University of Fez B.P.: 1223, Taza-Gare. Taza, Morocco. 2Laboratory of Human Pathologies Biology, Department of Biology, Faculty of Sciences, and Genomic Center of Human Pathologies, Faculty of Medicine and Pharmacy, Mohammed V University in Rabat, Morocco. 3Laboratory of Materials, Natural Substances, Environment and Modeling (LMSNEM), Polydisciplinary Faculty of Taza, Sidi Mohamed Ben Abdellah University of Fez B.P.: 1223, Taza-Gare. Taza, Morocco. *Corresponding Author E-mail : benali.taoufiq@gmail.com

Since antiquity, plants form an important source of metabolite secondary to develop remedies to treat various diseases. 1 Recently, several scientific researchers have demonstrated that natural plant extracts possess biological properties such as antimicrobial, antioxidant, anti-diabetic, anticancer, and anti-inflammatory activities. [2][3][4][5][6][7] Cistus Ladaniferus growing naturally in the Mediterranean region, namely in Morocco "Touzalt; touza'ch" frequently used in traditional medicines to treat many pathology such as stomachic problems and low libido case. 8,9 Several studies of the chemical composition of Cistus Ladaniferus essential oil (CLEO) indicate that its main compounds were viridiflorol, p-cymene, camphene, bornyl acetate, pinocarveol, a-pinene, and ledol. 10 Another work identified some flavonoids in the leaf of C. Ladaniferus. 11 In addition, a high content of viridiflorol, blumenol, limonene and spathulenol are detected in C. Ladaniferus methanol and ethanol extracts. 12 The biological effects of Cistus Ladaniferus as antibacterial, antioxidant, antifungal, and cytotoxic effects of C. Ladaniferus antifungal, are documented by several scientific works. [13][14][15][16][17] Mentha Suaveolens (Lamiaceae family) locally known as "m'chichro" is traditionally used to treat cold, digestive system ailment, hemorrhoid, fever, intestinal swelling, flu against chill, and as carminative and conception and fertility booster. 8,9,18,19 The genus Mentha is largely distributed in temperate regions of the world. 20 Mentha Suaveolens was characterized by free flavonoid. 21 Mentha Suaveolens essential oils (MSEO) from different regions were characterized by high levels of menthone, piperitone oxide, pulegone, isomenthone, and piperitenone oxide. 22 The oils of many Mentha species exhibit an important biological proprieties. [23][24][25][26][27] This study aims to determine the phenol and flavonoid contents of C. Ladaniferus and M. Suaveolens extracts and to evaluate the antioxidant and antimicrobial activity of these extracts, CLEO and MSEO.

Plant Material
Cistus

solvent extracts
Dried powder (10g/100 mL) of aerial part of the plant was macerated with ethanol, methanol, and water at ambient temperatures. The extracts were filtered and concentrated using a rotary evaporator to obtain a dry extract; for aqueous extract the water was eliminated with the freezedrying apparatus. All extracts were stored at 4-8°C until antimicrobial test was assessed.

estimation of total Phenolic and Flavonoid Contents
In this work, TPC was estimated using the Folin-Ciocalteu method 28 . The Folin-Ciocalteu reagent 1/10 with methanol was prepared which 2.5 mL of this solution was mixed with 0.5 mL of a sample solution. Then 4 mL of sodium carbonate (Na2CO3) (7.5 %) was added. After 30 min of incubation in water bath at 45°C the absorbance was measured at 765nm. TPC was expressed as mg of gallic acid equivalents per g of extract dry weight (mg GAE/g of extract).
The total flavonoid contents (TFC) in the extracts were revealed using the aluminum chloride method. 29 In brief, 0.3 mL of 5% sodium nitrite (NaNO2) was mixed with 5mL containing dissolved sample and distilled water. After 5 min, 0.3 mL of 10% aluminum chloride (AlCl3) was added and the solution was allowed to stand for 6 min. Then, 2 mL of 1 M sodium hydroxide (NaOH) was added and to reach 10 mL the volume was completed with distilled water. The solution was mixed vigorously and allowed to stand for 30 min. The absorbance was measured at 510 nm. The flavonoid content was determined as the rutin equivalent from the calibration curve of rutin standard solutions and expressed as rutin equivalent (mg RE/g of extract).

antioxidant activity Free radical scavenging activity
The radical effect of EOs and extracts was evaluated using the radical 2.2-diphenyl-1picrylhydrazyl (DPPH) as reported by Huang et al.30, with some modifications. Briefly, the DPPH solution (0.2 mM in methanol) was prepared. Then 2.5 mL of test sample at different concentrations (2.5-100 µg/mL) were added to 0.5 mL of DPPH solution and the absorbance of samples was measured at 517 nm after 30 min. Ascorbic acid and Trolox were used as positive controls.
Ascorbic acid and Trolox were used as positive controls. The calculation of the antioxidant activity was done according to the formula: DPPH scavenging activity (%) = [(A0 -As) / A0] * 100 Where A0 is the Absorbance of the negative control and As is Absorbance of the test sample at 30 min. The test was carried out in triplicate and the IC 50 values were reported as means ± SD. reducing power of ferric ions The reducing activity of EOs and extracts was determined according to the method of Oyaizu.31 The mixture of the 1 ml the phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and the potassium ferricyanide (2.5 mL) was prepared. After incubation for 20 min at 50 °C (water bath), 2.5 mL of trichloroacetic acid (10%) was added to mixture. Then, the solution was centrifuged at 3000Trs/min for 10 min. Finally, 2.5 mL of the supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride. Absorbance was measured at 700 nm.
Ascorbic acid (50-450 µg/mL) is used as a standard. The reducing power is expressed in milligram equivalence of ascorbic acid per gram of Essential oil (mg AAE/g of EO).

antimicrobial activity Microorganism strains and Growth Conditions
To study the antibacterial activity of plant extracts, we have used the following bacteria Listeria innocua CECT 4030, Staphylococcus aureus CECT 976, Bacillus subtilis DSM 6633, Proteus mirabilis, Escherichia coli K12, Pseudomonas aeruginosa CECT 118, Candida albicans ATCC 10231 was the fungi used to evaluate the antifungal activity.
The pathogen bacterial strains were cultivated in Mueller-Hinton agar (MHA) or Mueller-Hinton Broth (MHB) at 37 °C for 24 h. The fungi were cultured in YPGA medium (5 g yeast extract, 5 g Peptone, 10 g Glucose, 15-18 g Agar, in 1liter) or YPG, and incubated as follows: 48 h at 30°C for fungi;. The inoculum test concentrations are 106 CFU/mL for bacteria and 105 spores/mL for fungi.

agar disc diffusion Method
T h e a n t i m i c r o b i a l a c t i v i t y o f C. Ladaniferus and M. Suaveolens extracts and EOs was performed using the agar disc diffusion method.32 Briefly, sterile discs (6 mm diameter) containing 12.5 µL of pure essential oil or 20 µL of ethanolic, methanolic, and aqueous extracts at a concentration of 60 mg/mL dissolved by dimethylsulfoxide (DMSO) 10% were applied onto the surface of the agar-medium which was previously seeded by spreading by the inoculum concentrations test. After incubation, as described above, inhibitory zones were measured. Streptomycin 25 µg and DMSO 10% were used as positive and negative control, respectively.

Minimum inhibitory Concentration (MiC) and Minimum Bactericidal Concentration
The MIC values were determined in sterile 96-well microplate as described by Güllüce et al. 20, with some modifications. First, 100 µL of YPG was distributed in all test wells, except the first well in which a volume of 200 µL containing the essential oil or extracts with a concentration of 25 mg/mL in 10% DMSO. A series of concentrations ranging from 0.097 to 25 mg/mL were prepared by the transfer of 100 µL by scalar dilutions from the first to the ninth well. Then, except the 10th well used as sterility control, 10 µL of the suspension from each well was removed and replaced by the inoculum test concentration as described above. The eleventh well was considered as positive growth control containing only YPG broth. The last well containing 10% DMSO (v/v), without extracts, was used as negative control. Then, the plates were incubated at conditions of growth as described above. After an incubation, a volume of 25 µL of an indicator of microorganism's growth the was added in each well, the tetrazolium [MTT: 3-(4,5-dimethythiazol) -2-yl-2, 5-diphenyltetrazolium bromide (Sigma)] prepared at a concentration of 0.5 mg/mL in sterile distilled water. The microplate was re-incubated for 30 min at temperature 25°C. Where microbial growth was inhibited, the solution keeps the initial color of MTT. To determine the minimum bactericidal concentration (MBC) value, 10 µL of broth from the uncolored wells was inoculated in LPGA and incubated for 24 h or 72h at 25°C.

results estimation of Phenol and Flavonoid Contents
The estimation of phenol and flavonoid contents are expressed as mg of gallic acid or rutin equivalent per gram, respectively, was presented in Table 1. For C.Ladaniferus, The results showed that the aqueous extract is richer on TPC (76.98 ± 4.6 mg GAE / g of extract) compared to the methanol extract (59.8 ± 4.99 mg GAE/g of extract) and the ethanol extract (53.5 ± 6.28 mg GAE/g extract).
However, TFC are only present the aqueous extract (35.51 ± 2.04 mg ER/g of extract). Concerning M. Suaveolens, the methanolic extract showed a high TPC with value of 112.04 ± 2.98 mg GAE/g of extract), followed by the ethanolic extract whose content is in the range of 101.07 ± 4.34 mg GAE/g extract, while 92.70 ± 5.82 mg GAE/g extract are found in the aqueous extract. Furthermore, the content of the methanolic extract in flavonoids and of the order of 99.77 ± 5.4 mg RE/g of extract, followed by a content of 89.02 ± 4.23 mg RE/g of aqueous extract, on the other hand the content found in the ethanolic extract is 68.66 ± 2.94 mg RE/g of extract.

antioxidant activity
The results of antiradical activity of C. Ladaniferus and M. Suaveolens were summarized in Table 1. As we can see all extracts exhibit an anti-DPPH in a manner of dose-dependent ( Fig.  1 and 2). For C. Ladaniferus, the results showed that the aqueous extract exhibits a significant antiradical effect (IC 50 = 2.93 ± 0.02µg/mL), followed by methanolic extract (IC50 = 12.09 ± 0.02µg/mL), and the ethanolic extract reduces the DPPH with IC 50 value of 26.06 ± 0.05µg / mL. However, a low anti-DPPH effect was observed with MSEO with IC50 value superior to 100µg/ mL. For M. Suaveolens the results showed that the most important anti-free radical activity is caused by the aqueous extract IC50 = 8.78 ± 0.02µg/ mL, followed by ethanol extract IC50 = 12.57 ± 0.03µg/mL, and methanol extract IC50 = 15.83 ± 0.05µg/mL.
The results of FRAP assay, expressed as mg EAA/g, are summarized in Table 1. For C. Ladaniferus the aqueous extract possesses a significant reducing power of 39.53 ± 6.04 mg EAA/g of extract, followed by the methanolic extract with a reducing power of 23.69 ± 0.48 mg EAA/g of extract, and the ethanolic extract with 21.08 ± 0.06 mg EAA / g of extract. One the other hand, among the extracts of M. Suaveolens, the methanolic and aqueous extract have significantly (p <0.05) a very significant reducing power 54.44 ± 5.52 mg EAA / g of extract and 53.03 ± 1.65 mg EAA/g of extract, respectively, compared to the ethanolic extract 22.45 mg EAA/g of extract.   Table 2     The present study indicates that the extracts of C. Ladaniferus are characterized by a high content of phenol content. These results are similar to previous studies that report a high phenolic content. 33 Contrary to our results, Amensour et al. 12 reported that the methanolic and ethanolic extracts of C. Ladaniferus have a lower phenol content of 18.43 ± 2.74 mg GAE/g of extract and 11.87 ± 0.53 mg GAE/g of extract. Concerning M. Suaveolens, the high phenol contents of this plant were previously reported. 34 Compared with plants of the genus Mentha, the results of other study indicates that the methanolic and aqueous extracts of the species Mentha spicata, Mentha pulegium and Mentha piperita are rich in phenol with contents which vary between 167.2 ± 0.41 and 305.4 ± 1.32µg TAE/mg including aqueous extract of Mentha spicata contains the highest phenol contents. 35 The quantitative variation on the phenol contents may be attributed to some parameters such as the chemical structure, the method of extraction, the size of the particles forming the sample, and the solvent. 36,37 There is evidence in the literature that that the antioxidant activity correlates with the phenolic contents. 38,39 Our results showed a dose-dependent percentage inhibition of DPPH reduction and a high reducing power of extracts of Cistus Ladaniferus in order aqueous>methanol>ethanol extracts; these results may be attributed to the presence of high phenol content in these extracts. Interesting antioxidant results are obtained for M .Suaveolens. Indeed, the aqueous extract showed the most significant anti-free radical activity, followed by the ethanol and methanol extract; knowing that the phenol-rich extracts are in the order methanol> ethanol> aqueous. The missing correlation between the phenol content and antioxidant activity could be explained by numerous factors; (i) the presence of non-phenolic products such as sugars, amino acids which reacts with Folin-Ciocalteu giving a high apparent phenolic level, 40,41 (ii) the antagonistic interaction between phenolic and non-phenolic compounds, 42 (iii) the antioxidant activity depends on the numbers and position of functional groups. 43 For the antimicrobial activity; on the hand, the C. Suaveolens extract and essential oil were active against both gram-positive and gramnegative bacteria. The absence of antibacterial selectivity effect may be attributed to the presence of high content of products known for this propriety in the chemical composition of essential oil, and to the high phenol content in crude extracts. On the other hand, the gram positive strains were most sensitive to the essential oil of M. Suaveolens compared to the gram negative bacteria, these results may be attributed to the presence of phospholipids and lipopolysaccharides in Gramnegative membrane protected them against the external environment. 44

ConClusion
This study has displayed that extract of C. Ladaniferus and M. suaveole was possessed antioxidant and antimicrobial activities. Accordingly, we may need more achievement works in future to determine the specific active components which, are responsible for this antioxidant and microbial activity with suggestions for a mechanism of actions.

aCknowledGMent
We are very thankful to the Laboratory of Biology and Health, Sciences Faculty of Tetouan and the Laboratory of Agri-Food and Health of FST-Settat, for providing us with the microorganisms used in this study.