Phytochemical Investigation and Biological Activities of Some Saharan Plants from Hoggar

Traditional medicine has a great impact on aboriginal heal thin Algerian Sahara and particularly in Hoggar. It is based on the use of local plants. This study is based on phytochemical screening. Antioxidant activity evaluation by two methods of 1,1-diphenyl-2-picrylhydrazyl (DPPH): bioautography and spectrophotometric assay, and microbiological activity of twelve plants species from nine families sampled in Hoggar. The antioxidant activity revealed by the bioautography (DPPH) showed a positive activity on aqueous extracts of ten species among the twelve assayed. Both species Lavandula pubescens subesp. antinea and Mentha longifolia (Lamiaceae) have reported a significant activity compared to the others. The comparison of antiradical activity bybioautography and phytochemical study by TLC showed the presence of cardiotonic glycosides, saponins and flavonoids which could be responsible of antioxidant activity or some species. The antimicrobial activity assay showed that plant aqueous extracts were inefficient on the majority of tested strains except for Balanites aegyptica extract on Bacillus subtilis ATCC6633. Moreover, remarkable antifungal activity of Aerva javanica aqueous extracts on four fungal strains such as Candida albicans C200, Aspergillus flavus AF1, Aspergillus carbonarius M333, Fusarium culmorum FC1was observed.


Introduction
Plants use for therapeutic purpose was reported in the ancient Arab literatures.In Africa, the therapeutic plants action was known by our ancestors and our parents in an empirical way (Karou et al., 2003).Until now, both medicines: traditional and modern are practiced.To achieve a better knowledge of this African medicine, several phytochemical investigations have been made in order to bring a justification and a scientific validation to its traditional use (Yemoa et al., 2008).Many work undertaken in ethnopharmacology field, showed that the used plants in traditional medicine which were tested are often considered as effective plants in the pharmacological models on the other hand, they would be almost deprived of toxicity.
The antimicrobial activity was treated by De Oliveira et al. (2011) for Brazilian plants and Al-Bayati (2009) for Iraqi ones.
Several works were carried out in Algeria on plants of medicinal interest.These plants were evaluated for their richness in polyphenols and their antioxidant, antimicrobial, and antidiabetic proprieties (Djeridane et al., 2006).Zaabat et al. (2010) conducted a phytochemical study on Marrubium desertii.He isolated an original diterpene of labdane type, a flavonoïd and a glycosyled phenyl propanoïdes.These compounds were subjected to biological study as antioxidant, antibacterial and antigenotoxic activities.
The work carried out on fifty-six species of medicinal plants harvested in different areas of Algeria showed the richness of secondary metabolites and biological activity like the antioxidant and the acetyl cholinesterase activity (Benamar et al., 2010;Rached et al., 2010).
Despite many studies carried on biological activity of prospected plants in Algeria, many species remain still not investigated.Indeed, central Sahara hosts vegetal species resistant to extreme aridity conditions.This natural richness indicated various foods, cosmetic and medicinal virtues (Sahki et al., 2004).Until now, traditional healers belonging toTuareg tribes use these plants for treating several diseases.In this context, our investigation was conducted to evaluate the antioxidant and antimicrobial activities of twelve Hoggar plants species.

Biotope of Studied Plants
The Ahaggar (Hoggar) is an integral part of the central Sahara subdivision; it is constituted by mountain range rising to over 3000 m of altitude.It is located at the extreme south of Algeria and was approximately between 21°-25° North latitude and between 3°-6° West longitude (Gast, 1968).It covers a surface of approximately 554,000 Km², which represents the quarter of Algeria total surface (Sahki, 2012).The Ahagar flora is characterized by vegetal formations, especially tropical and saharo-sindian, which is concentrated mainly in Oued's bank and dry valley at middle and low altitudes.On the contrary, higher altitudes are less arid, then host many microclimates allowing mainly Mediterranean, endemic and endemic relic flora development.
Since ancient era, Tuaregs (inhabitants of Ahaggar) were interested by plants curative virtues growing in their natural environment.Generally, this traditional culture was transmitted by nomadic and semi-sedentary population, despite of sciences progress.
Ethnomedicinal surveys conducted with traditional healers's Ahaggar reported that different plant parts (leaves, roots, fruits, bark, seeds, flowers, wood) are used directly in the fresh or powder form, mixed with other plants, tea, honey or animal fats.Many obtained preparations were generally administered in the form of teas, baths, syrup or extract added to food or drink (Sahki, 1998).

Plant Material
The studied sample is represented by aerial parts of fifteen plants harvested from Hoggar area in May, 2009 and February, 2010 (Table 1).These plants were identified by Dr. Sahki, member of INRF (National Institute of Forest Research) and then were put in herbarium (Sahki et al., 2004).The samples were dried at free air in the darkness or in a drying oven at 50 °C during 24 h and then crushed to form a fine powder.

Lavandula pubescenssubsp. antinea
Leaves stems and flower Infusion used against the rheumatism and the spasms.These is antiseptic of the respiratory and urinary tracts.These is used as a condiment and flavoring.

Marrubium desertii
Leaves buds Decoction used against bloating and diarrhea, they have efficient avirtues against tachycardia

Mentha longifolia
Leaves, flowers and fruits The infusions of fresh leaves are digestives, stomachic and calming.The flowers and fruits are astringent infusion.The leaves are used as condiment infusions

Stems, leaves flowers and Roots
Used in decoction, they relieve kidney's pain, intestinal disorders and food poisoning; they used in rheumatic and sinusitis.It treats the wounds of dromedary by cataplasm

Balanites aegyptiaca
Fruits, barks, roots, leaves, oils extracts by almond Decoction of the bark is used to relieve throat.Fruit relieve sinusitis, tonsillitis and protect the face against the cold.The leaves are laxative, digestive, anthelmintic, relieve rheumatic pain, nausea, boils and dental caries.The oily paste extracted from the almond of the fruits used in cosmetics and for treating scabies dromedaries

Extraction
The samples were extracted by heat under reflux, 10 g of plant powder were put in100 ml of distilled water.This operation is repeated three times during half an hour.The three obtained filtrated were mixed, lyophilized and stored at -4 °C until further use.

Solution Preparation
The freeze-dried powder was weighed and dissolved in methanol at the concentration of 5 mg/ml for all the samples.

Phytochemical Screening by TLC
The phytochemical analyses of the plant extracts were carried out following the methods of (Wagner & Bladt, 1996), the chromatographic analyses were achieved out on plates of CCM silica gel F 254 (Merck).Several systems were used, the chromatograms are observed under U.V at 254 and 365 nm.After revelation, the detected phytoconstituants are: flavonoids, saponins, terpenoïds, glycosides cardiotonics, sesquiterpenes lactones, quinones and lignans.

Total Phenolic Content Determination
The total phenolic content is estimated using Folin-Ciocalteu's method (Singleton & Rossi, 1965).100 µl of sample was dissolved in 500 µl of Folin-Ciocalteu reagent (1/10 dilution).The solution were mixed and incubated at room temperature during 5 mn.Then, 1500 µl of saturated sodium carbonate (2%) was added.The final mixture was mixed and then incubated for one hour in the dark at room temperature.The absorbance was read at 765 nm.

Total Flavonoid Content Determination
Total flavonoid contents were determined by the method of (Kim et al., 2003).500 µl of the methanolic samples were added to 1500 µl of distilled water and 150 µlof sodium nitrite (NaNO 2 ) at 5% (m/v).After 5 min, 150 µl of aluminum chloride (AlCl 3 ) at 10% (m/v) was added.11 minutes later was added 500 µl of 1 M NaOH.The solution was vortexed then the absorbance was measured at 510 nm.
2.4.4Total Tannin Content Determination 100 µl of methanolic sample extract in pure methanol was added to 600 µl vanillin at 4% in absolute methanol and 300 µL of concentrated HCI (Burns et al., 1971).Incubation is conducted in water bath at 30 °C during 20 min.All the samples were maintained in the dark.The absorbance is read at 500 nm, we used as a control, a solution containing methanol instead of the vanillin solution.

Antiradical Activity Determination by Thin Layer Chromatography (TLC) (Bioautography)
The 2,2-diphenyl-1-picryl hydrazyle (DPPH) method was used to determine antiradical activity according to (Dominguez et al., 2005).The antioxidant test is carried on thin layer chromatography, 100 µg of plant extract are deposited on aluminum plates of silica F 254 .The chromatograms are developed in a mixture of several solvents.After separation and drying, the plates are pulverized with a 0.2% solution of DPPH in methanol.

Test DPPH by Spectrophotometry Dosage
The method of (Blois, 1958) modified by (Rached, 2010) includes only extracts which presented antiradical activity revealed by bioautography.The test is carried out according to the following stages: the methanolic solution of DPPH (1950 µl) at a concentration of 6.10 -5 M was mixed with 50 µl of different extract concentration (5-10-12.5-15-20µg/ml).Then, the mixture was incubated at room temperature in darkness for 1 h.The absorbance of each extract containing DPPH was read at 517 nm using a UV-visible spectrophotometer (8500P Double-Beam spectrophotometer).The percentage of inhibition (PI) of the extract is calculated according to the equation: ( Where, A neg. control is the absorption of the negative control solution (containing only DPPH); A sample is the absorption in the presence of the plant extract in DPPH solution; Quercetin and the BHA (Butyl Hydroxy Anisole) are used as the positive controls.
The experiment is repeated three times for each sample and the value is expressed by an average ± standard of deviation.

The Antimicrobial Activity Evaluation
The antibacterial and antifungic activities of extracts were investigated.

1) The Bacterial and Antifungal Strains Tested
The strains of Bacillus subtilis ATCC6633, Staphylococcus aureus, Escherichia coli E195, Klebsiella pneumoniae E40, Candida albicans C200, Aspergillus flavus AF1, Aspergillus carbonarius M333, Fusarium culmorum FC1 were sampled from Bejaïa and Algiers hospitals, they were provided by the laboratory of microbiology of the (ENS).

2) Determination of Antimicrobial Activity
Antibacterial activity of the aqueous extract of the sample was evaluated by paper discs diffusion method.The stock concentration was prepared from the aqueous extracts of the tested plants at a concentration of 200 mg/ml.After that, sterile filter papers (9 mm diameter) were impregnated with 5 mg and 10 mg of extract by disc.All disks prepared were then dried in air and sterilized under UV.
The impregnated discs by various extracts are then deposited delicately on the surface of the agar medium.The antibacterial activity was determined by measurement of inhibition zone around each paper disc.Incubation was done at 30 °C during 18 h to 24 h for bacteria strains and 36 h to 48 h for fungi and yeast strains.

Statistical Analysis
The results are expressed on average ± standard deviation.ANOVA (variance analysis) compare several groups by using the Duncan test.For comparison between two groups, the student test was used.The values were significant at P < 0.05.

Phytochimical Result by TLC
Phytochimical study results of the tested vegetal extract were indicated in Table 2.The orange, yellow, blue, green spots observed on the chromatogram under UV/365 nm correspond to several secondary metabolites classes.The chromatograms showed a wealth of metabolites for some extracts.A comparison is made in parallel between the phytochimical and TL Cbioautography study by DPPH in order to focus on the active phytoconstituants.

Total Polyphenols, Flavonoids and Tannins Determination
The polyphenol content of the studied extracts has 230.003±8.423mg range for Lavandula antinea leaves and 33.635±0.477mg of gallic acid equivalent per gram of freeze-dried matter in Balanites (aerial parts).The flavonoïds content vary between 94.483±4.137and 9.537±0.657mg of catechin equivalent per gram of freeze-dried matter of Lavandula antinea (stems) and Balanites aegyptiaca (aerial parts).Tannins are present in 21.36±0.33mgrange for Solenostemma argel leaves and 3.3±0.92mg of catechin equivalent per gram of matter freeze-dried in Mentha longifolia stems.
Lavandula antinea (stems) given an IC 50 value of 10.352 ± 0.388.This value was determined in comparison to the reference value of IC 50 BHA which is estimated to 4.457±0.07.

Antioxidant Activity by Bioautography
The free radicals activity of fifteen aqueous extracts were investigated.The obtained results showed that thirteen extracts reacted with the DPPH, illustrated by the appearance of yellow bands on a purple background.Some of these extracts had appeared instantaneously but others appeared after 30 minutes.

DPPH Dosage Assay
The absorbance measurement (optical density) was conducted by a spectrophotometer at 514 nm.From the obtained values, the percentage of inhibition was calculated by using the formula the formula given in one methodology part.Note.*Antioxidant activity expressed as µg ml -1 .**Total phenolics contents expressed as mg gallic acid in g of freeze-dried extract.*** flavonoids and tannins contents as mg catechin g -1 of freeze-dried extract.nd: Not determined, +: Positive result, -: Negative results.Data expressed as Mean±SD from triplicate experiments.BHA: Butyl-hydroxyanisole.

Antimicrobial Activity Determination
The antibacterial and antifungal activities of the various extracts were estimated by inhibition zone diameter evaluation after 12 h and 24 h incubation at 30 °C temperature for two concentrations 5 mg/ml and 10 mg/ml.

1) Inhibition Zone Diameter for 5 mg/ml Concentration
After 24 hours of incubation, inhibition zones of 12 mm are observed on only 04 extracts concerning gram positive bacterial Bacillus subtilis.After 48 hour, significant inhibition is noticed for Aspergillus flavus (Tables 3 & 4).2) Inhibition Zone Diameter for 10 mg/ml Concentration Concerning E. coli E195 and Staphylococcus aureus, strains, the results were negative for all the tested extracts.
The results of inhibition zone diameter for Bacillus subtilis, showed that this strains sensitive to Pentzia monodiana, Lavandula antinea and Pulicaria incise extracts.Nevertheless, these extracts did not develop any inhibition zone for gram negative bacteria, yeasts and fungi.
Mentha longifolia and Aerva javanica extracts at 25 μl (5 mg/ml) concentration showed a significant inhibition of Aspergillus flavusfungus with 13 mm diameter.For 50 μl concentration, results were not significant for all organisms and sample.The Balanites aegyptiaca extract shown the most important inhibition zone for Bacillus subtilis (Table 5).

Discussion
According to spots intensity and number, the most antioxidant extracts are those of Lavandula antinea (stems and leaves), Mentha longifolia (leaves), Pentzia monodiana and Deverra scoparia.This antioxidant activity may be explained by the presence of tannins and flavonoïds (Bruneton, 1993;Cavin, 1999a), which are free radicals trappers.Cavin (1999b) demonstrated that aqueous extract of Lamiaceae had less antioxidant activity by TLC, with unclear spots, especially for aqueous extract of Thymus vulgaris because of their low active components levels that may be due to the method extraction used (extraction by maceration with water).Whereas, the methanolic extract of Thymus vulgaris showed the most capacity trapper of radical DPPH.
Moreover, among the aqueous extracts assayed, Maerua crassifolia presented the lowest antioxidant activity.These results were consistent with those of (Diallo, 2005) with the aqueous extract macerated leaves of the same plant (data not published).
Our results reported that the DPPH reduction by spectrophotometric assay showed that Lavandula antinea extracts (stems and leaves) had a higher antioxidant capacity in comparison with BHA control; this activity might be attributed to the phenolic compounds richness.Similar data were observed with the aqueous extract of Thymus vulgaris leaves (Kulšic et al., 2006).In this extract, polyphenols such as flavonoids, acid rosmarinic, caffeic acid and α-tocopherol can explain the revealed activity (Guillén & Manzanos, 1998;Thuille et al., 2003;Kulšic et al., 2006).The less significant activity compared to BHA was obtained with Balanites aegyptiaca.For the same plant, the antioxidant activity is variable from one organ to another (Wagner & Bladt, 1996).In our investigation, Lamiaceae species did not present this variability which is only noted for Marribium desetii, its leaves had low activity and its roots do not show any activity.The isolated compounds (diterpens) from the arial part of this Algerian endemic species presented antiradical, antibacterial and antigenotoxic activities (Zaabat et al., 2010).Our phytochimic study revealed richness in some compounds and the lack even absence of other ones.Some species had shown the presence of saponosids and the cardiotonic glycosides.Flavonoids were the most common compounds in all studied species.Markham et al. (1982) showed that anthocyanins-3-glycosides where detected by orange, red and purple spots, while, flavonols by yellow and green spots.Flavones methylate, flavones, isoflavones, flavanones and chalcones appeared as blue spots.The flavanols and the aurones are characterized by the appearance of the green spots.
A comparative study between phytochimical and antioxidant activity by DPPH TLC showed that some bands represent the same R f for some species, this explained the reason that flavonoïds, saponins and cardiotonic glycosides had antioxidant activity.Flavonoïds are recognized to have antioxidant activity (Torel et al., 1986;Husain et al., 1987;Shahidi & Wanasundara, 1992;Harborne & Williams, 2000;D'abrosca et al., 2007).The present study indicated that the antimicrobial activity was significant at 10 mg of extract.There was a sensitivity of gram positive bacterium Bacillussubtilis to the majority of studied extracts.This could be explained by the fact that gram-positive bacteria was more sensitive than gram-negative bacteria, suggesting that difference in sensitivity was caused by repulsion between polyphenols and lipopolysaccharide walls of gram negative bacteria.Generally, the sensitivity of bacteria against polyphenols depends on bacterial species and polyphenols structure (Ikigai et al., 1998).
Our findings showed that aqueous extracts fromMentha longifolia manifested high antifungal activity in leaves as compared to stems, but no activity was observed towards yeast.Such results could be explained by a difference in concentration of chemicals between both vegetal parts.Al-Bayati ( 2009) reported antifungal activity with different concentrations of Mentha longifolia essential oil on the majority of seven bacteria studied with a strong antifungal activity against Candida albicans.The richness of polyphenols and flavonoïds of Lavandula antinea extract did not indicate any antifungal and antibacterial activity.All plants of Lamiaceae family, known for their phenolic compounds, were proven as active against variety of micro-organisms (Gortzi et al., 2007).Aerva javanica appears as an interesting plant, it showed an antifungal activity against Candida albican sand all tested yeasts with considerable inhibition zones.We noted that aqueous extracts have a low antimicrobial activity.The comparative work between alcoholic and aqueous extracts showed that C. alata, L. camara and M. scaber alcoholic extracts produced a more significant inhibition zones than the aqueous ones at a 500 mg/ml concentration (Ali-Emmanuel et al., 2002).It could be stated from this analysis that each extract acts differently on micro-organisms.A given compound can have a significant effect on one germ or less or no effect on another.
Marcelline Adiko et al. (2014) tested some plants were harvested in their natural habitats.Aqueous and methanol extracts of dried material were screened for antimicrobial activity, by dilution in solid culture medium, against six bacterial species responsible for ocular infections.Seven extracts were active against all bacteria methanol extracts of Hibiscus asper (Malvaceae), Hoslundia opposita (Lamiaceae), O. gratissimum, S. mombin, Vitellariaparadoxa (Sapotaceae) and Z. zanthoxyloides (bark); aqueous extracts of Piliostigma thonningii (Fabaceae) and S. mombin.

Conclusion
This work revealed the richness of medicinal flora from extreme southern Algeria which is traditionally used in the treatment of several diseases.Our phytochemical and biological activitie studies have been focused on the aerial parts, leaves and stems.The phytochemical analysis revealed the presence of saponins, cardiac glycoside sand flavonoids.Some tested extracts have the capacity to scavenge free radicals.Antibacterial, antifungal, and anti-oxidant activities were demonstrated for certain plants Lavandula antinea (stems and leaves), Mentha longifolia (leaves), Pentzia monodiana, Deverra scoparia and Aerva javanica.

Table 2 represents
IC 50 values (corresponding to 50% of inhibition) of the extracts and represents the antioxidant activities.The aqueous extract of Lavandula antinea (stems) given an IC 50 value of 10.352±0.388.This value was determined in comparison to the reference value of IC 50 BHA which is estimated to 4.457±0.07.

Table 2 .
Chemical composition of studied plants

Table 3 .
Inhibition zone diameter of active plants extracts against Bacillus subtilis strain at 5mg concentration

Table 4 .
Inhibition zone diameter of active plants extracts against Aspergillus flavus at 5 mg concentration

Table 5 .
Inhibition zone diameter of active plants extracts at 10 mg concentration