INTRODUCTION:

Pinus halepensis Miller considers one of the most important medical plants which belongs to pinaceae family, this plant commonly known as Aleppo pine, it's native region is Mediterranean and it's distributed from Morocco, Algeria, Albania, Spain, France, Croatia, Montenegro, Italy, east to Greece and all over Mata and northern Tunisia [1].

Aleppo pine is a minor to moderate sized tree, the high is reached to about 15-25 m, the leaves are needles and very slender, definitely yellowish green and formed in pairs [2][3].

The cones are fine 5-12cm long and 2-3cm wide at the base when closed, green at first maturing smooth and brown when 24 month old. They exposed gradually over the following few years, a development accelerated if they are exposed to hotness such as in forest fires [4]. The cones open 5-8cm wide to permit the seeds to separate, the seeds are 5-6mm long with a 20mm wing and are wind spread [5]. Pine cone extract has a medical history that times retain to the Greek physician Dioscorides (A.D 514). In recent day, Japanese used it as a normal therapy to treat disease and illness ranging from the communal cold to cancer [6]. Pine cone extracts contain a large number of chemical constituents containing polysaccharides, lignins, tannins and some types of phenolic acids. In Iraq there are non or only a few studies about cones of this plants, therefore this study was carried out to evaluate the phytochemical constituents of cone of P. halepensis grown naturally in the north of Iraq and to estimate the antimicrobial and antioxidant activity of its extracts.

MATERIALS AND METHODS

Plant materials:

Cones of P. halepensis were obtained from Sulaimania in the north of Iraq, then classified by Iraqi National Herbium in Abu-Ghraib. The cones were washed by tap water to remove dirt and dust and rinsed with distilled water then dried at room temperature for 10 days then crushed into well powder.

Preparation of plants extract:

10gm of the cone powder of Aleppo pine were placed in thimble and extracted in a soxhlet apparatus by 250 ml of hexane for 96hr, then the extract was filtered by Whatman filter paper No.1. The filtrated extract was dried under vacuum using rotary evaporator (Heidolph Germany) Lipophilic extract [LE]. The mark remain in the thimble and extracted with 250ml (acetone/water 9:1) for 96hr in the soxhlet apparatus then the extract was filtered using Whatman filter paper No.1. The filtrated extract was evaporated under vacuum to dryness by rotary evaporator, hydrophilic extract [HE]. Another 10g of the cone powder of Aleppo pine were extracted with 200ml ethanol using soxhlet apparatus for 72hr, then the extract was filtered by Whatman filter paper No. 1 then evaporated under vacuum to dryness by rotary evaporator, ethanolic extract[TE].

Phytochemical Analysis

Qualitative phytochemical analysis was carried out by using different standard methods in order to investigate the secondary metabolites presented in the ethanolic extracts of Aleppo pine .

Tannins

A few milligrams of ethanolic extract was diluted with water, 3-4 drops of 10% ferric chloride solution is added. A green color is detected for catecholic tannins while a blue color designates for gallic tannins.

Saponins

2.5ml of ethanol extract was added to 10ml of sterile distilled water in a test tube. The test tube was closed tightly and shacked strongly for about 30 seconds and permitted to stand for 30 min. Honeycomb froth designate the exist of saponins.

Flavonoids

2ml of ethanolic KOH was added to 1ml of ethanolic extract, yellow color indicate the presence of flavonoids.

Glycosides

25ml of diluted HCl was added to 5ml ethanolic extract and heated for 45 min, cooled and neutralized with 10% NaOH, then 5ml Fehling solution is added. A black red sediment designate the presence of glycosides.

Terpenoids

4ml of the plant extract was treated with 0.5ml of acetic anhydride and 0.5ml of chloroform. Then concerted solution of sulphuric acid was added gradually and red violet color was seen for terpenoids.

Sterols and steroids

Liebermann reaction were used to indicate the presence of steroids. 10ml of ethanolic extract was vaporized. The residues was dissolved in 0.5ml of hot acetic anhydride and filtered. 0.5ml of the filtrate was treated with Libermann burrchardt. The appearance of a blue-green ring at the interphase showed a positive result.

Coumarins

0.5ml 10% NH4OH was added to 5ml of ethanolic extract. On a filter paper 2 spots was added and examined under U.V light. Intense fluorescence indicates the presence of coumarins.

Alkaloids

Extracts were dissolved individually in diluted hydrochloric acid and filtered. The filtrate was separated into two parts, the first part was treated with Wagner's reagent (iodine in potassium iodide). Development of brown/reddish sediment designates the existence of alkaloids.

The second part were treated with Dragendroff's reagent (solution of potassium bismuth iodide). Development of red sediment designates the existence of alkaloids.

Collection of microorganisms and preparation of stock culture:

Clinical microorganisms were received from Department of Biology, College of Science, University of Baghdad and definite by gram staining and cultured in suitable selective media.

Table 1:Microorganisms used in the study

Gm+vebavteria	Gm –ve bacteria	yeast
Staphylococcus aureus	Escherichia coli	Candida albicans
Streptococcus pneumonia
α-hemolytic Strep. viridans

Estimation of antimicrobial activity:

The three extracts (HE,LE,TE) of cone extract of Aleppo pine were dissolved in methanol to obtain a final concentration (100,50,25 mg/mL) and sterilized using 0.22 Millipore filter. Antimicrobial estimation was done using agar well diffusion method. The stock culture of microorganisms were refreshed by streaking using a sterile inoculation loop on nutrient agar medium plates in a laminar flow hood, then incubated at 37^οC for 24 hrs., then the inoculate were diluted in a sterile saline solution to a total concentration of 10⁷ colony forming units (cfu/mL) modified to 0.5 McFarland standard solution. The diluted bacteria then spread on a Muller-Hinton agar (Oxoid U.K), the Muller-Hinton agar were punched using six diameter wells and filled with (100, 50, 25) mg/mL of each extracts. Solvent (methanol) was used as a negative control whereas ciprofloxacin (10µg/disc) was used as a positive control. Plates were incubated at 37^οC for 24hr, then the diameter of the zone of inhibition around the well was measured in mm and documented for the extracts the plant.

Antioxidant activity (DPPH free radical scavenging):-

Antioxidant activity of extracts (LE,HE,TE) of cone extract were measured using the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH).In a test tube 0.5 mg/mL of each extract was mixed with 5ml (100µM) freshly prepared DPPH-methanol solution and the reaction mixture was set aside in the dark at room temperature for 30 minute. There after the absorbance was recorded at 517nm. DPPH react with the antioxidant extract of the plant and get reduced to DPPH-H form thereby decreasing in absorbance value . The percentage inhibition of DPPH radical was measured by matching the results of the test samples with those of the control (not treated with extract) using the following equation:-

Percentage inhibition =(1- absorbance of test/absorbance of control) x 100

RESULTS AND DISCUSSION:

Plant extracts preparation

Three solvents were used to extract Aleppo pine. The results of 10g for each extract were: Hexane extract was yield Lipophilic extract [LE] 300mg. Acetone/water (9:1) extract was yield hydrophilic extract [HE] 1.1g. Ethanol extract was yield of ethanolic extract [TE] 1.6g.

Phytochemical Investigation

Table (2) shows the results of chemical testes.

Table 2: Qualitative phytochemical analysis of the ethanolic extracts of cone of P.halepensis

Secondary metabolite	Results
Saponins	-ve
glycosides	+ve
alkaloids	-ve
flavonoids	+ve
terpens	++ve
coumarins	-ve
Tannins	-ve
Sterol and steroids	-ve

-=not detected; +=slight coloration; ++= deep coloration

This study showed that cone extracts have a good antimicrobial activity against Gm+ve, Gm-ve bacteria and yeast as compared with the broad spectrum antibiotic ciporofloxacin (10µg/disc) as shown in tables 3, 4, 5. the hydrophilic extract gave higher zone of inhibition against Gm-ve E. coli (25mm) at the conctration 25mg/mL, while lipophilic extract gave higher zone of inhibition against Gm+ve Staph. aureus (24mm), TE extract gave the best antimicrobial activity against all strains. The antimicrobial activity of this plant maybe to different active compounds, such as glycosides, flavonoids and specially phenolic terpenes which have antiseptic activity, and these results agreement with other studies were refereed to secondary metabolites [8].

P. halepensis may show an important role in plant series from side to side several processes. For example, secondary compounds (terpenoids and/or phenolic compounds) can affect root symbionts and site quality, by intervening with mineralization, decomposition and humification [9-10]. P. halepensis may inhibit seedling formation of numerous species in pine stands, proposing the allelopathic nature of litter, leaf leachates, and/or root exudates [11-12].

Table 3: Zone of inhibition in mm of HE of cone of P.halepensis

Microorganisms	Conc. of extract in mg/ml			Ciprofloxacin (10µg/disc)
Microorganisms	100	50	25	Ciprofloxacin (10µg/disc)
Staph. aureus	20	21	22	18
Strep. pneumonia	15	20	20	21
α- Strep. viridans	22	22	17	25
Escherichia coli	-----	20	25	16
Candida albicans	16	17	22	18

Table 4: Zone of inhibition in mm of LE of cone ofP.halepensis

Microorganisms	Conc. Of extract in mg/ml			Ciprofloxacin (10µg/disc)
Microorganisms	100	50	25	Ciprofloxacin (10µg/disc)
Staph. aureus	17	24	15	18
Strep. pneumonia	13	15	16	21
α- Strep. viridans	20	18	15	25
Escherichia coli	19	14	14	16
Candida albicans	15	20	14	18

Table 5: Zone of inhibition in mm of TEof cone of P. halepensis

Microorganisms	Conc. Of extract in mg/ml			Ciprofloxacin (10µg/disc)
Microorganisms	100	50	25	Ciprofloxacin (10µg/disc)
Staph. aureus	20	21	19	18
Strep. pneumonia	17	13	13	21
α- Strep. viridans	20	18	17	25
Escherichia coli	11	16	13	16
Candida albicans	15	12	13	18

The antioxidant activity of P. halepensis is very good, good and poor for hydrophilic, ethanolic and lipophilic extract respectively as compared with ascorbic acid table 4, figure 2. This activity maybe to the working of terpenoides which pull free radicals, and antioxidant factors [7], and this effect may be due to the containment terpenoides [8] and flavonoides that are effective vehicles antioxidant [13].

P. halepensis cones showed significant antioxidant activities as well as high levels of bioactive compounds. Therefore, P. halepensis cones are a potential resource of natural antioxidants that may give some health interests. Though, the lipid extract of P. halepensis cones seem to have more nutritious value as a polyunsaturated oil than that of PHC that is high in saturated and trans fatty acid [14] .

Table 6: Antioxidant activity 0.5 mg of each extracts as compared with ascorbic acid

	Cone extract of P. halepensis			Ascorbic acid
	TE	HE	LE	Ascorbic acid
Absorbance at 517	0.457	0.058	0.724	0.23
%DPPH scavenging activity	55	94	28	77

Figure 2: Comparison of scavenging activity between different extract of cone of P. halepensis.

ACKNOWLEDGMENT:

The authors would like to thank Mustansiriyah University(www.uomustansiriyah.edu.iq) Baghdad- Iraq for its support in the present work.

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Received on 06.06.2018 Modified on 12.07.2018

Research J. Pharm. and Tech 2018; 11(11): 4977-4980.

DOI: 10.5958/0974-360X.2018.00907.1