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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Chouikh et al.

World Journal of Pharmacy and Pharmaceutical Sciences SJIF Impact Factor 5.210

Volume 4, Issue 11, 337-349

Research Article

ISSN 2278 – 4357

PHYTOCHEMICALS STUDY, ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF HELIANTHEMUM LIPPII (L.) PERS. IN DIFFERENT STAGES OF GROWTH (SOMATIC, FLOWERING AND FRUITING). Chouikh Atef1, 2*, Feriani Anouar3, Adjal El Hadda1, Chefrour Azzedine2,4 1

Department of Biology, Faculty of Natural Science and Life, University of El Oued, BP 789 El-Oued (39000). Algeria. 2

Laboratory Development and Control of Hospital Pharmaceutical Preparations. Medical Science Faculty, University of Badji Mokhtar- Annaba, Algeria. 3

4

Lab of Ecophysiologie Animal, Faculty of Sciences, University of Sfax, Tunisia.

Faculty of Natural Sciences and life, University of Mohamed Cherif Messaadia-Souk Ahras (41000) Algeria. ABSTRACT

Article Received on 17 Oct 2015, Revised on 19 Oct 2015, Accepted on 22 Oct 2015

Objective: The aims of this work were to study the differences in amount of phenolics compounds and to estimate the antioxidant, antimicrobial activities of Helianthemum lippii (L.) with respect to growth stages (Somatic, Flowering and Fruiting). Methods: Three

*Correspondence for

methanol extracts of different growth stages were prepared to

Author

estimation of phytochemicals content and evaluate the antioxidant and

Chouikh Atef

antimicrobial activities. Results: Yields of E.MOH extract is low

Department of Biology, Faculty of Natural Science

during fruiting (6.5%) compared with the flowering period (8.97%)

and Life, University of El

and the Somatic period (8.24%). The quantitative estimation of

Oued, BP 789 El-Oued

polyphenols and flavonoids showed that the extracts are rich in these

(39000). Algeria.

compounds. Antioxidant activities showed that the methanol extract of the Somatic stage has a significant efficacy on DPPH radicals and

hydroxyl, with the extract against methanol fruiting looks best on the free radical peroxide. As regards the extract from the flowering stage, it has been less significant results on all three radicals. The bacterial strains showed a very significant sensitivity via-a-via the extract of the flowering stage followed by fruiting, while a net resistance is observed with the extract of the

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World Journal of Pharmacy and Pharmaceutical Sciences

Somatic stage. Conclusions: one can deduce that the introduction of this pastoral plant in the medical field would be desirable to enhance the therapeutic arsenal in the medical world. KEYWORDS: Helianthemum lippii (L.) Pers., Methanol extracts, Phytochemicals, Antioxidant activity, Antimicrobial activity, Stages of growth (Somatic, Flowering and Fruiting). 1. INTRODUCTION The genus Helianthemum belonging to the family of Cistaceae[1] contains 08 genera and 200 species[1,2,3] which are widespread in regions Mediterranean.[3] Across the world, this genus includes 70 species[4] In Algeria and Pakistan, this genus has a single species Helianthemum lippii (L.) Pers.[3,5] The vernacular name of this species is different in different regions and continents, for example the name of Al Samhari (in the region of Oued Souf : Southeast of Algeria)[6]; Reguig (in the region of Ouargla: South of Algeria) Tahsowat and Alrjik (South-West of Algeria); Alrkaroq (in Kuwait), Umm Souika (the Arabian Peninsula)[7] and Sun Flower (Northeast of Jordan).[8] It is a perennial herbaceous plant [6,9] Hard Evergreen, Small, heights ranging from 10 to 45 cm[10] and can reach up to 50 cm.[11] Its stems grouped into a few white branches[11] A small leaves[6] does not exceed a length of 01 cm; the often small yellow flowers located on one side of the inflorescence. Flowering in late spring, however, the flowers appariaient in different seasons[6] and can be pollinated by bees Apis mellifera L.[8] In point of view Ecological and economic, this plant is very interesting [6, 9]

pastoral plants

[9]

It is part of the

, it plays an important role in the fight against desertification and contribute

to the stability areas threatened by desertification. The powder of the species used to treat skin rashes[9] It is also used in Libya against rot and rashes and prevent ailments.[12,13] In Morocco, this plant with the Bedouins can cause lameness in camels in the name of GAF or Kraft (a type of arthritis). But in reality, the toxicity of this plant has not yet proven.[14] To our knowledge there are no reports about influence of different growth stages (Somatic, Flowering and Fruiting) on phytochemical content and antioxidant, antimicrobial activities of

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methanol extracts of Helianthemum lippii (L.) Pers. So, the goal of this work was to study the differences in amount of phenolic compounds and to estimate the antioxidant, antimicrobial activities of Helianthemum lippii (L.) with respect to growth stages (Somatic, Flowering and Fruiting). 2. MATERIAL AND METHODS 2.1. Preparation of plant material The plant material consists of the aerial part of the plant Helianthemum lippii (L.) Pers.; harvested in Oued Souf Sahara (South East of Algeria) during three stages of plant development (Somatic stage in February 2010, the flowering stage in March 2010, at fruiting stage month of April 2010). The plant is then dried, crushed and stored in glass bottles shielded from light and humidity. 2.2. Chemicals The Methanol purchased from Biochem Chemophara ( Montreal, Quebec, Canada); All the chemicals used in this study were of analytical grade, 2,2_-diphenyl-1-picryl-hydrazyl (DPPH), Folin-Ciocalteu reagent, Gallic acid, Quercetin, Tannic acid and Ascorbic acid (Vitamin C) were purchased from Sigma Chemicals Co (St. Louis, MO, USA). 2.3. Preparation of methanol extracts 20g dry matter herbal drugs are impregnated each in 200 ml of absolute methanol for 24 hours. After filtration, the methanol solutions are evaporated to dryness under reduced pressure in a rotary evaporator kind Buchi R-200 at 55 ° C[15] The yield of the plant methanolic extract is determined by the formula. Yield (%) = (P1 / P2) x 100. 

P1: weight of the methanol extract dried in gram.



P2: weight of the plant starting material in gram.

2.4.Quantitative analysis: 2.4.1. Content of total polyphenols Total phenol are determined according to the method described by[16] 0.1 ml of the methanol extract was mixed with 0.9 ml of Folin-Ciocalteu reagent (1N). Then 0.2 ml of a solution of Na2CO3 (20%) is added to the mixture. The mixture was incubated at room temperature,

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protected from light, for about 40 minutes. The absorbance is measured at 725 nm. The results are expressed in mg equivalent Gallic acid (0.02 to 0.12 mg / ml) / g DM). 2.4.2. Dosage of flavonoids The flavonoids content was determined using as reference the Quercetin. A volume of 250 μl of methanol extract and 1 ml of distilled water are mixed. At time t 0 is added 75 μl of sodium nitrate (NaNO3, 5%), after 5 minutes is added 75 μl of AlCl3 (5%). After 6 minutes a volume of 500 μl of NaOH (1N) is added. Absorbance is read at 510 nm. The flavonoid content is expressed in mg Quercetin equivalents / g DM.[17] 2.4.3. Dosage of condensed tannins Mixing 50 μl of methanol extract with 1.5 μl of vanillin (4%), then add 750 μl of HCl (5%). The mixture is incubated for 20 minutes at room temperature. The absorbance is read at 500 nm. The results are expressed in mg Tannic acid equivalents/g DM.[18] 2.5. Antioxidants Tests Highlighting the antioxidant activity in vitro Helianthemum lippii (L.) Pers. is carried out by three test namely trapping free radical DPPH, hydroxyl radical scavenging (OH •) and trapping of hydrogen peroxide (H2O2). 2.5.1. Test sequestration free radical DPPH The antioxidant activity of different extracts was measured by the method described by [19] with a slight modification. Then we proceeded to the preparation of a range of dilutions (20 to 120 μg / ml) from the mother solution methanolic extracts of H. lippii (L.) Pers. Then a series of tubes is prepared as follows. • Sample (Extract + DPPH): 1ml of each dilution of the methanol extract + 1 ml of DPPH (4 mg / 100 ml methanol). • Control: 1 ml of distilled water + 1ml DPPH. The tubes are incubated at 37 ° C until a yellow coloring. The absorbance is measured at 515 nm. Percent inhibition is determined by the following formula: I % = [(Abs control - Abs sample) / Abs control] x 100. Abs sample: absorbance of all the mixture. Abs control: absorbance of the sample without mixing.

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2.5.2. Scavenging test of Hydrogen Peroxide (H2O2) Trapping free radicals of peroxide was determined by the method of[20] with some modifications.1ml of methanolic extracts of H. lippii (L.) Pers. mixed with 1.2 ml H2O2 (6 mM) in a weakly basic medium (phosphate buffer 0.1 M, pH 7.4). The sample is incubated for 10 minutes and the absorbance was measured at 230 nm. The percentage of inhibition of peroxide was determined by the following formula. I (%) = [(Abs control - Abs sample) / Abs control] x 100 Abs sample: absorbance of all the mixture. Abs control: absorbance of the sample without mixing. 2.5.3. Test scavenging Hydroxyl (OH•) The hydroxyl radical scavenging test is measured by the method described by[21] with some modifications. The mixture was prepared with 1 ml methanolic extracts of H. lippii (L.) Pers., 1ml FeSO4 (1.5 mM), 0.3 ml of Salicylic acid (20 mM), 0.7ml H 2O2 (6 mM). The mixture is incubated for 60 minutes at room temperature. Absorbance is read at 510 nm. Percent inhibition (I %) is calculated by the following formula: I % = [1 - (Abs sample / Abs control)] × 100 Abs sample: absorbance of all the mixture. Abs control: absorbance of the sample without mixing. 2.5.4. IC50 determination. This parameter is defined as corresponding to the concentration of antioxidant necessary to decrease the initial concentration of 50 %, it is inversely related to antioxidant capacity. Over the IC50 value is smaller, the antioxidant activity of a compound is high.[22] 2.6.

antimicrobial activity

The antimicrobial activity of methanol extracts H. lippii (L.) Pers. was determined by agar diffusion method cited by[23] Sterile disks (6 mm diameter) impregnated with 100 μl of methanol of H. lippii (L.) Pers. extracted three concentrations (250 μg / ml ; 500 μg / ml and 1000 μg / ml) are then deposited on the agar surface previously seeded with bacterial suspension (106 CFU / ml) in exponential growth phase. The Petri dishes were incubated at 37° C for 24 h. The inhibition of microbial growth is determined by measuring the inhibition diameter (mm) of each bacterium. [24]

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3. RESULTS AND DISCUSSION 3.1.Yield of methanol extracts. The yield of the methanol extracts (fig.01) is low during fruiting (6.5% ± 0.74%) compared to the other two phases of growth of the plant (flowering, 8.97% ± 1.44%, Somatic, 8.24 ± 1.16%).

Fig.01. Yield methanol extracts of H. lippii (L.) Pers in different stages of growth (Somatic, flowering and fruiting). 3.2. Contents of polyphenols substances Spectrophotometry was possible to assay the rate of polyphenols in the methanol extract of H. lippii (L.) Pers. The concentrations of polyphenols, flavonoids and condensed tannins were determined from calibration lines (regression line) using, respectively, as the standard Gallic acid, Quercetin and Tannic acid. 

y = 20,92 x + 0,477; R² = 0,913.



y = 0,87 x + 0,019; R² = 0,986.



y = 0,917 x - 0,007; R² = 0,974.

The concentration is expressed in milligrams of Gallic acid equivalents per gram of dry matter (mg GA E / g DM) for the polyphenols, Quercitin milligram equivalents per gram of dry matter (mg QU E / g DM) for flavonoids and milligrams of Tannic acid per gram of dry matter (mg TA E / g DM) for the condensed tannins. Table (01) illustrates the richness of H. lippii (L.) Pers. in active substances. During the three phases of plant development, the values of phenolic compounds are almost identical except for the Somatic stage (1.389 ± 0,1065 mg GA E / g DM). The flavonoids rate dropped to a

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very minor way during the three phases. Against it was noticed by the presence of screw to screw fluctuation tannins content. The rate in the Somatic stage of 0.9425 ± 0.0895 mg TA E / g DM, followed by a reduction in flowering (0.3595 ± 0.046 mg TA E / g DM) and another peak is obtained fruiting (0.536 ± 0.0785 mg TA E / g DM). Table 01. Quantification of Phenolic compounds. phases of growth of H. lippii (L.) Pers. Total polyphenols (mg GA E / g DM) Flavonoids (mg QU E / g DM) Tannins (mg TA E/ g DM) 3.3.

Somatic 1,389 ± 0,1065 0,9425 ± 0,0895 0,638 ± 0,1

flowering 1,294 ± 0,0385 0,9345 ± 0,1615 0,3595 ± 0,046

fruiting 1,298 ± 0,1215 0,931 ± 0,211 0,536 ± 0,0785

Antioxidants Tests

3.3.1. Reducing activity of DPPH In this context, the methanol extract of Somatic stage has better antioxidant capacity against the radical DPPH (IC50 58.98 ± 0.70 μg / ml). This value is far from those of the Ascorbic acid which is a standard antioxidant (IC50 34.99 ± 0.90 μg / ml). 3.3.2. Reducing activity of the Hydroxyl radical (OH •) The most effective concentrations (IC50) used to inhibit 50% of the hydroxyl radical are respectively of the order of 324.84 ± 44,29 μg / ml of the Somatic stage extract, 401.32 ± 51.51 μg / ml of the flowering stage extract and 341.09 ± 4.29 μg / ml of the extract fruiting stage. These values are more or less small compared to the use of Ascorbic acid regarded as a standard antioxidant (224.67 ± 3.44 μg / ml). 3.3.3. Reducing activity of hydrogen peroxide (H2O2) The effective concentration (IC50) determined from the curve is of the order of 392.95 ± 48,51 μg / ml (Somatic stage); 464.48 ± 45,50 μg / ml (Flowering stage); 345.30 ± 21,35 μg / ml (Fruiting stage), the latter (Fruiting stage) is close to Ascorbic acid which is a standard antioxidant (303.18 μg / ml). 3.3.4. The IC50 of antioxidants tested The IC50 values for Ascorbic acid (Vit C) and methanolic extracts H. lippii (L.) Pers. are indicated in the table (02) From these results it is proved that the ascorbic acid remains the most effective antioxidant with an IC50 (34.99 μg / ml in free radical DPPH ; 303.18 μg / ml to free radicals of Peroxide H2O2 ; 224.67 μg / ml to Hydroxyl radical OH •) from the methanol extracts of H. lippii (L.) Pers. for the three phases of development of the plant. The

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methanol extract of the Somatic stage is the thread manager for its antioxidant capacity against radical DPPH and Hydroxyl, while the extract of the Fruiting stage gave the best results against free radical Peroxide compared to first two stages of growth. Table 02: IC50 values of methanol extracts of H. lippii (L.) Pers. against free radicals (DPPH; Peroxide H2O2; Hydroxyl OH•).

extracts Helianthemum lippii (L.) Pers. Standard

Stages of growths

DPPH (IC50; µg/ml)

Scavenging of Free Radicals Peroxide (IC50; µg/ml)

Somatic Flowering Fruiting Ascorbic acid (Vit C)

58,98 ± 0,70 61,49 ± 2,33 60,68 ± 8,00 34,99 ± 0.90

392,95 ± 48,51 464,48 ± 45,50 345,30 ± 21,35 303,18 ± 23.62

Scavenging of Free Radicals Hydroxyl (IC50; µg/ml) 324,84 ± 44,29 401,32 ± 51,51 341,09 ± 4,29 224,67± 14.22

3.4. The anti bacterial and anti fungal activity The results of the anti microbial activity of methanol extracts H. lippii (L.) Pers. are summarized in table (03). Table 03: anti bacterial and anti fungal activity of different concentrations of methanol extracts of H. lippii (L.) Pers. in different phases of growth (Somatic, Flowering and Fruiting).

phases of growth Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus Klebsiella pneumoniea Candida albicans

Somatic Flowering Fruiting C1 C2 C3 C1 C2 C3 C1 C2 C3 (250 (500 (1000 (250 (500 (1000 (250 (500 (1000 µg/ml) µg/ml) µg/ml) µg/ml) µg/ml) µg/ml) µg/ml) µg/ml) µg/ml) 7

15

24

0

17

29

0

7

7

0

0

0

17

20

20

17

18

25

18

18

21

0

12

17

13

14

25

0

20

20

16

17

20

0

0

16

11

12

14

0

8

15

7

7

12

The comparison of the activities of methanolic extracts H. lippii (L.) Pers. In three stages reveals a high sensitivity of the tested germs except for E. coli which showed resistance with different concentrations of the methanolic extract of the Somatic phase. P. aeruginosa germ is very sensitive to high concentrations of the first two stages of plant development. As against E. coli parrait very sensitive to different concentrations of the last

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two phases of development (Flowering and Fruiting), this is probably due to the production of a bactericidal substance by the plant during flowering and fruiting. S. aureus are sensitive to all concentrations aside the dose (250 µg / ml of methanolic extract obtained from the Flowering phase. The K. pneumoniea showed vis-a-vis resistance concentration 250µg / ml of the Somatic phase and the first two levels of the Fruiting stage. But it is sensitive to all concentrations of various phases of growth of the plant. The most important anti fungal effect is observed with the highest concentrations of the two first phases of development of the plant. We can deduce that the methanol extract of the Flowering stage is very efficient (high values of inhibition zones), followed by the extract of the Fruiting period, while the extract of the Somatic stage is not nearly motionless on E. coli. 4.

DISCUSSION

The increases in yield during flowering is probably due to pollination phenomena and embryogenesis, followed by a slight decrease during the fruiting because of the degradation of these active ingredients and use them as a reserve for stores the new off spring.[25, 26] The volatility observed in the values of multi-phenols quantity, flavonoids and tannins through the three stages of growth (Somatic, Flowering and Fruiting) due to: a difference phenolic composition of the samples studied[27] and due to the nature of the soil and local climate (microclimate) in the study sites.[28, 29] The volatility in yields alcoholic extract values through stages of growth is due to the difference growth phase.[30] As to external conditions such as the environment and the soil a big role in it.[29, 30] The value (IC50 of DPPH) are relatively low with those obtained by[13] working on the H. lippii (L.) Pers. methanolic extract of Libya on this free radical (IC 50 31.17 ± 1.40 μg / ml). Plant that contain high amounts of polyphenols not necessarily give dynamism large antioxidant[31]; The effectiveness of anti-oxidation are closely linked to the structure and

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quality of phenolic compounds than the concentration and quantity of these compounds within the plant tissue.[32] The strong effect of anti-oxidants in some samples of alcoholic extract of the plant H. lippii (L.) Pers. during different growth stages due to the as proposed Benhammou [28] to the possible presence of compounds type of Flavanols Such as: Kaempferol and Quercétine which effectively isolated from the plant Helianthemum glomeratum by[33]; It also explains the difference in antioxidant activity between samples for different behavior to give a proton and an electron between samples.[34] Our results of activity antimicrobials are consistent with those obtained by[13] who were able to show that methanolic extracted from the same plant in their countries (Libya) have good antimicrobial activity against gram-positive bacteria (S. aureus with inhibition zone 21 mm) and the fungal strain Candida albicans with a zone of inhibition of 20 mm, but no effect on gram-negative bacteria (E. coli). CONCLUSION Phytochemical study, antioxidants and antimicrobial methanol extracts of a pastoral plant Helianthemum lippii (L.) Pers. collected in the region of Oued Souf (North East Algerian Sahara) during different stages of growth (Somatic, flowering, fruiting) revealed that: 

The yield of methanol extract is low during the fruiting compared to Somatic and Flowering stages; this is probably due to the conversion of some active substances reserves of products to be accumulated in the storage organs.



The increase in the yield of active substances (phenolic compounds) during the flowering period has a close relationship with the pollination and pollinators vectors of this species.



The quantification of Phenolic compounds of three periods gives values close except Tannins Who were low in Flowering stage compared to other stages of growth.



The methanol extract of the Somatic phase has a better antioxidant effect against the radical DPPH and hydroxyl groups. In return, the fruiting phase produces highly effective active substances against free radical peroxide over the two growth stages.



Depending on results, we can say that the methanol extract of the Flowering stage gives large areas of inhibition followed by the extract of H. lippii (L.) Pers. the Fruiting phase.

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At the end of this work, one can deduce that the introduction of this pastoral plant in the medical field, namely totem or its extracts would be desirable to enhance the therapeutic arsenal in the medical world. REFERENCES 1. Kubitzki K, Bayer C. The families and genera of vascular plants. Berlin; SpringerVerlag., 2003. 2. Guzman B, Vargas P. Historical biogeography and character evolution of Cistaceae (Malvales) based on analysis of plastid rbcL and trnL-trnF sequences. Organisms Diversity & Evolution, 2009; 9(2): 83-99. 3. Perveen A, Qaiser M. Pollen Flora of Pakistan Cistaceae. Karashi; University Karashi., 1998. 4. Sambamurty AVSS. Taxanomy of angiosperms. New Delhi; Chand and Company Ltd., 2005. 5. Ozenda P. Flore du Sahara. 2ème éd., Paris; CNRS., 1977. 6. Halis Y. Plant Encyclopedia in area Oued Souf: desert plants common in the Big East race. El oued Algeria; El walid., 2007. 7. Mandaville J P. Flora of eastern Saoudi Arabia. London; Springer., 1950. 8. Zaitoun ST, Vorwohl G. Major Pollen Plant Species in Relation to Honeybees’ Activity in the Jordanian Desert Area. Paris; Taylor et Francis group., 2003. 9. Hamza A, Gtari M, Neffati M. Micropropagation of Helianthemum lippii L. var Sessiliforium (Cistaceae) an important pastoral plant of North African arid areas. African Journal of Biotechnology, 2013; 12(46): 6468-6473. 10. Hamza A, Hamrouni L, Hanana M, Hamza F, Gtari M, Neffati M. In vitro Micropropagation of Helianthemum lippii L.var Sessiliforuim (Cistaceae): A Valuable Pastoral Plant Middle-East. Journal of Scientific Research, 2012; 11(5): 652-655. 11. Quezel S, Santa S. Nouvelle flore de l’Algérie et des régions désertique méridionales. Paris; CNRS., 1963. 12. Ermeli NB, Alsabri SG, Bensaber SM, Mohamed SB, Zetrini AA, Aburas KM, et al. Screening of analgesic and anti-inflammatory activities for two Libyan medicinal plants: Helianthemum lippii and Launaea residifolia. Journal of Chemical and Pharmaceutical Research., 2012; 4(9): 4201-4205. 13. Alsabri SG, Rmeli NB, Zetrini AA, Mohamed SB, Meshri MI, Aburas KM, Bensaber SM, Mrema IA, Mosbah AA, Allahresh KA, Hermann A, Gbaj A. Phytochemical, anti-

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