Harvest date and variability in lipid bioactive compounds in Pistacia atlantica

Abstract

BACKGROUND:

The present work stands as an endeavor to uncover the ideal harvesting time of leaves in which they exhibits the maximum contents of bioactive molecules such as essential fatty acids, tocopherols and carotenoids.

METHODS:

A large scale investigation was carried out for the leaves of Pistacia atlantica involving a large number of populations collected over a period of four months during the growing season. The antioxidant activity was evaluated using both DPPH and β-carotene assays. The chemical percentage variability of the fatty acids was investigated using statistical analysis methods (Agglomerative Hierarchical Clustering “AHC”, also cited as CAH).

OBJECTIVE:

During the growth period, the effects of harvesting date on the total contents of lipids, tocopherols, carotenoids, fatty acids composition and also the antioxidant activities of the lipids were investigated.

RESULTS:

The content of myristic acid and other saturated fatty acids increased during leaf development, while linoleic, linolenic acids and unsaturated fatty acids decreased. The highest percentages of both linolenic C18:3 (27.25±5.92%) and linoleic acids C18:2 (17.68±3.80%) were obtained for the month of May at the first stage of leaves development (young leaves), but higher percentage levels of C18:1, were obtained for both consecutive months of August & September (28.83±6.50%; 27.79±8.63%, respectively) at intermediate developing stage. The lipids, tocopherols, carotenoids contents and the saturated and unsaturated fatty acids were dependent on the harvest time. The antioxidant activity showed higher powers at the first developing stage (May). Two main clusters and two sub-clusters of the fatty acids were distinguished and were also depending clearly on the period of the collection.

CONCLUSIONS:

The results showed that the FA were dependent on the period of collection of the leaves. The main result of this study illustrate the nutritional potential (richness in MUFA such as C18:1, 2, 3) of the oil of P. atlantica leaves, which can provide opportunities for rational exploitation in the food industries or for medicinal purposes.

Keywords

leaves fatty acids lipids antioxidant activity development stage

1 Introduction

One of the objectives of the food industry is to seek new resources of oil that preferably presents nutritional values. For human health, fats and particularly vegetable oils are considered as an important source of energy when glucose is not available. Lipids play an important role in food and nutrition serving as a source of essential fatty acids, vitamins, carotenoids and antioxidants compounds. As part of lipids, unsaturated fatty acids UFA are known for their multiple dietary, pharmaceutical and clinical benefits. The naturally occurring fatty acid: conjugated linoleic acid (C18:2), presents different properties such as anti-atherogenic and anti-carcinogenic [1]. In the same context and besides its role in reducing cardiovascular disease risk, studies evaluated the direct anti-atherogenic and anti-inflammatory effects of dietary α-linolenic acid (C18:3) polyunsaturated fatty acids (PUFA) in addition to their lipid-lowering effects [1, 2]. Vitamin E in the form of tocopherols or tocotrienols is an example of natural antioxidants found in lipids fractions [3]. Antioxidants serve as protective role by scavenging free radicals such as reactive oxygen species that can lead to the damage of cell membrane. Carotenoids are also of commercial significance due their application as food colorants.

In recent years, ethnobotanical studies performed in several countries indicated that Pistacia species play a vital role in the nutrition and agricultural economy of many communities, living in arid and semi-arid regions [4, 5]. The Pistacia nuts are edible, with abundance in unsaturated fatty and other bioactive compounds. For their nutritional profile, nuts have been reported to reduce the risk of coronary heart disease, blood pressure, cancer, inflammation, gallstones, and diabetes [4]. Pistachio acquired its economic importance from being one of the most favored nuts around the world. Moreover, pistachio has a high oil content consisting of about 50–62% of its weight [6]. Pistachio nut particularly is rich in protein, dietary fiber, potassium, magnesium, vitamin K, γ-tocopherol, and a number of phytochemicals. With renewed interest in relevance of ethnobotanical uses, several properties such as antioxidant, wound healing, antimicrobial, antiviral, anticholinesterase, anti-inflammatory, antinociceptive, antidiabetic, antitumor, antiancer, antihyperlipidemic, antiatherosclerotic, and hepatoprotective have been evaluated [4]. Ongoing studies on pistachio extracts has demonstrated lately the confirmed effects of its kernel on cellular viability, intracellular reactive oxygen species (ROS) production and cell death in MCF-7 breast cancer cells [7]. Although pistachio oils have different nutritional values according to the nut origin source, pistachio cold pressed oil has generally a great amount of unsaturated fatty acids and bioactive phytochemicals such as phenolics, stilbenes, carotenoids, phytosterols, and proanthocyanidins [6, 8]. Because of its special chemical composition, pistachio oil plays an important role in the industrial production of cosmetics, with positive gentle effects on skin and hair. Moreover, it can contribute significantly to the pharmaceuticals industry, as it is rich in bioactive ingredients, having both antioxidant and anti-inflammatory properties [6].

The Pistacia atlantica plant belongs to the Anacardiaceae family. Its origins are from the Mediterranean and the Middle East countries. The French botanist R. L. Desfontaines discovered it in 1798 [9]. This plant grows in the Algerian steppe and desert areas. It grows in clean forest. The Pistacia atlantica tree has significant importance as follows: it resists the high temperature and it does not need much water; the Pistacia atlantica tree protects the steppe region and humid the areas because it is drought resistant; it preserves the soil, stabilizes and cools it.

The Pistacia atalantica tree produces a huge quantity of fruits, leaves and galls utilized traditionally for culinary and medicinal purposes and not yet used in industrial applications. All these organs of the tree are rich in natural bioactive compounds. P. atlantica is considered a functional food, because it is rich on nutrients, including sugars, fatty acids, amino acids, minerals, vitamins, and antioxidants [5]. The fruits contain an important amount of oil with predominance of unsaturated fatty acids (73–83%) [10 –12], and also the oil is rich in tocopherol compounds [13]. Frequent fruit consumption has shown a reduced risk of cardiovascular disease and lowering effects of total and LDL cholesterols as well as properties to eliminate stomach diseases [14]. Hispolone and luteolin are naturals antioxidants that has been isolated from the fungus Inonotus hispidus growing on Pistacia atlantica [15]. The different organs of Pistacia atlantica tree contain the essential oil with predominance of monoterpene hydrocarbon [16 –23]. The essential oil of leaves of Pistacia atlantica is very important source of non-toxic natural antioxidants because of its significant antioxidant activity [24]. Also Pistacia atlantica leaves constitute a source of phenolic compounds with important antimicrobial, particularly antifungal, antioxidant, antidiabetic and antihypertensive [25]. The essential oil of the galls showed antimicrobial, antibacterial, antioxidant and low cytotoxic activities [26]. The amount of the phenolic content, the chemical composition of the essential oils of the Pistacia atlantica leaves and the antioxidants activities are dependent on seasons and vary the during stage of development of the leaves [22, 27].

Although the fatty acids composition, and the total contents of both tocopherols and carotenoids of Pistacia atlantica were recently reported by another team of our laboratory [28]. Nevertheless, this previous study was not conclusive since many parts were investigated at once (leaves, galls and fruits), using an extremely reduced number of samples (at number of four for each plant part). Actually, these samples were collected from the same location over three months of study period [28]. Upon these items, the variability factors were missing and hence the study results are considered preliminary (conclusions must be considered with precaution), and need to be further studied with more expanded regions and with larger number of samples over wider range of growth period.

Hence, this report aims to investigate at a large scale the change in the content in lipid fraction, the fatty acids composition, tocopherols, carotenoids content and the antioxidant activities of the lipids from leaves of Pistacia atlantica tree collected over an extended period of four months during the growing season and involving 22 different populations. Moreover, the antioxidant activitiy was determined using two different in vitro tests: DPPH and for the first time β-carotene bleaching assays (lipid peroxidation test). Data analysis using AHC was carried out in order to establish positive distinctive groups on the basis of the studied parameters. The present work stands as an endeavor to uncover the ideal harvesting time of leaves when rich, in bioactive molecules such as essential fatty acids, tocopherols and carotenoids.

2 Materials and methods

2.1 Chemicals

Absolute ethanol, methanol, n-hexane, chloroform, activated charcoal, sulfuric acid, acetic acid, ferric chloride, dichloromethane, Vitamin E and DPPH^• (1,1-diphenyl, 2-picrylhydrazyl) were obtained from Sigma-Aldrich (Saint-Louis, Missouri, USA). Anhydrous sodium sulphate and sodium were purchased from Analar NORMAPUR Prolabo (France). Butylated hydroxyanisole (BHA) was purchased from Fluka chemie (Buchs, Switzerland). Acetic anhydride was obtained from Merck (Darmstadt, Germany). Ortho-phenanthroline was obtained from Biochem Chemopharma (Cosne-Cours-sur-Loire, France). All reagents and chemicals used were of analytical grade.

2.2 Plant material

The study was performed on Pistacia atlantica leaves obtained from six different trees belonging to three sites namely: Site 1 (33°48’7^′′N and 2°53’19^′′E with an altitude of 760 m), site 2 (33°45’2^′′N and 2°48’06^′′E with an altitude of 777 m) and site 3 (33°39’14^′′N and 2°55’10^′′E with an altitude of 810 m). The six different chosen populations were picked-up randomly. The samples were collected at the bottom of each tree, and at different locations all around the tree. All samples were collected regularly during growth (different phenological states) at the beginning of each month for a period of four months (May “04 samples”, August “06 samples”, September “06 samples” and November “06 samples”, in year 2014). All leaf samples were green, except those obtained in November (autumn) which were in yellow color. In May, the leaves were young and still growing bigger. In full developed stage (August/September) the leaves were fully expanded. Finally at last stage (November) the leaves were tending to yellow color before falling.

Although, extraction of lipids should be performed as soon as possible after the removal of tissues from the living organism, sometimes, nonpolar solvents, such as diethyl ether and n-hexane, do not easily penetrate the moist tissues (>8% moisture); therefore, effective lipid extraction does not occur. Therefore, reducing moisture content of the samples may facilitate lipid extraction [29]. In this context, and since the solvent employed was n-hexane, the collected leaf samples of the plants were air-dried in the shade at room temperature for two weeks, prior to lipid extraction.

2.3 Lipid extraction

The air dried leaves of Pistacia atlantica were milled into powder using a coffee grinder. Lipid extraction was performed with n-hexane using a Soxhlet extraction apparatus for 2–3 days. The lipid extract was dried by adding 2 g of anhydrous sodium sulphate, filtered and evaporated to dryness using a rotary evaporator (temperature set at 40°C), with a ratio of 1:5 (mass) activated carbon to initially air-dried leaves, chlorophyll removal was carried out by adsorption on activated charcoal by reflux using chloroform as a solvent for 2 hours. The obtained extract was cooled to room temperature then filtered. Chloroform was removed from the solution on a rotary vacuum evaporator at 40°C. The lipid extract was weighed to determine the lipid content. The lipid fractions were subjected to another operation which is waxes removal. The lipid extract were dissolved in absolute ethanol and stored in the refrigerator for 24 hours, then filtered and evaporated at 40°C. The lipid extract was stored at+4°C for further analysis.

2.4 Fatty acids composition

In order to determine the fatty acids (FA) composition, lipids were transformed into fatty acid methyl esters (FAME), the method consisted of refluxing the lipids (0.5 g) within 20 mL of 0.5% sodium methylate (NaOMe prepared by action of sodium on methanol) for 25 min. After cooling, 20 mL of distilled water was added. In the next step, methyl esters of FA were extracted with dichloromethane (DCM), and then washed with distilled water. Subsequently, the obtained solution was dried over anhydrous sodium sulfate Na₂SO₄ then evaporated under vacuum at 40°C. Prior to gas chromatography (GC) analysis and identification, the FAME were purified by chromatography over silica gel as stationary phase and DCM was used as a mobile phase.

2.4.1 Gas chromatographic (GC) analysis of FAME

FAME were analyzed on a gas chromatograph (Chrompack, CP 9002, Netherlands) equipped with a split-splitless mode injection system, flame ionization detector (FID) and a DB23 capillary column (30 m×0.32 mm i.d., 0.32μm film thickness). Oven temperature was in isotherm mode (250°C). Injector and detector temperatures were set at 250°C. Nitrogen was used as carrier gas at a flow rate of 1 mL/min. The injection volume was 1μL. The fatty acids were identified by comparison of their retention times with those of pure standards.

2.4.2 Gas chromatography/mass spectroscopy (GC/MS)

FAME were analyzed by GC/MS using a Shimadzu GC/MS-QP2010 ultra equipped with Auto sampler AOC-20i, Ion source: electronic impact High-performance Quadrupole Mass Filter. Separation of compounds was carried out in a DB-5 ms capillary (Length 30 m; diameter 0.25 mm; film thickness 0.25μm) using helium as the carrier gas (99.99% purity) with a flow rate of 1.2 mL/min. The oven temperature was programmed at 70°C for 5 min, then 4°C/min to 250°C, followed by temperature rate, 2°C/min up to 300°C which was maintained for 5 min.

2.5 Total tocopherol content

The total tocopherol content (TTC) of lipid fractions was determined using the spectrometric method of Emmerie, et al. [30] with some modifications. In order to draw a calibration curve relating the optical density to the concentration, a series of known concentration solutions of commercial vitamin E was prepared in ethanol. 1 mL of 1,10-phenanthroline reagent (0.4% in absolute ethanol) and 0.5 mL of ferric chloride reagent (0.12% in absolute ethanol) were added to 1 mL of each solution. The mixture was incubated for 5 minutes at room temperature. Absorbance was measured at 510 nm (Shimadzu UV/Vis 1601 apparatus) against a blank prepared using 1 mL of ethanol, 1 mL of 1,10-phenanthroline reagent and 0.5 mL of iron chloride reagent. The dewaxed lipid samples were treated with the same procedure described above. TTC in dewaxed lipids were calculated from the vitamin E calibration curve and results were expressed as mg α-tocopherol eq./g of lipid. All determinations of TTC were carried out in triplicate.

2.6 Total carotenoid content

Determination of total carotenoids (TCC) of lipid fractions was performed by the method described by Rutz, et al. [31], with modifications. A standard calibration curve was obtained from different concentrations of β-carotene solutions prepared in dichloromethane; the absorbance of each solution was determined at 460 nm. Quantification of carotenoids in dewaxed lipids was carried out by the same procedure. The results were expressed as mg β-carotene eq/g of lipids. All determinations of TCC were carried out in triplicate.

2.7 In vitro antioxidant activity

2.7.1 DPPH radical scavenging assay

DPPH assay was performed using the stable radical 2,2-diphenyl-1-picrylhydrazyl as described by Molyneux [32], with a few modifications. Different volumes of dewaxed lipid dilutions in absolute ethanol were added to 1 mL of 100μM solution of DPPH^•. After 30 min of incubation at room temperature, the absorbance was read against a blank at 517 nm. The absorbance of the control was measured by replacing the sample with ethanol for each dilution volume. The percent scavenging activity I% was calculated as follows: $I % (Inhibition percentage %) = (1 - \frac{A_{sample}}{A_{blank}}) \times 100$

Where:

A_blank is the absorbance of the control reaction (DPPH with only ethanol) and A_sample is the absorbance in the presence of the extract sample.

The antiradical activity was expressed as EC₅₀ (mg/mL) which represents the concentration required to cause 50% initial DPPH^• inhibition. Commercial vitamin E and BHA were used as reference standards. All experiments were performed in triplicate.

2.7.2 β-carotene bleaching assay

The β-carotene bleaching method was established for each fraction trough the method proposed by Sun, et al. [33] with some modifications. 1 mg of β-carotene was dissolved in 10 mL of dichloromethane. 5 mL of β-carotene solution was mixed with 20 mg of linoleic acid and 200 mg of Tween 80, then dichloromethane was evaporated under vacuum at 40°C and the resulting mixture was immediately diluted with 50 mL of distilled water, then the emulsion was vigorously shaken. 40μL of different solutions concentrations were added to 1 mL of β-carotene/linoleic acid emulsion. The absorbance of the preparations was measured at 490 nm before and after 120 min of incubation in a water bath at 50°C against negative control prepared by substituting the extract with an equal volume of methanol. The antioxidant activity (AA%) was performed using the following equation: $AA % = [(\frac{A_{s 120} - A_{c 120}}{A_{c 0} - A_{c 120}})] \times 100$

Where:

A_s120 and A_c120 are the absorbance measured for the test sample and control, respectively, after 120 min of incubation.

A_c0 is the absorbance for the control measured at zero time before the incubation.

The results were also expressed by AA₅₀, the concentration providing 50% β-carotene bleaching inhibition). Vitamin E was used as positive control. All trials were performed in triplicate. All experiments were performed in triplicate.

2.8 Statistical analysis

2.8.1 Data analysis

Statistical analysis was carried out using XLSTAT Version 2016.02.27444. Experiments were conducted with six replicates (prior six randomly chosen tree populations). The results were expressed as mean±SD of three independent experiments (three leaves repetitions).

Raw data obtained were analyzed through analysis of variance (ANOVA) combined (as a posttest procedure) with Tukey’s post-hoc (HSD) multiple range tests. P-values of less than 5% (p < 0.05), were considered to be significant.

2.8.2 Cluster analysis

Cluster analysis was performed using Agglomerative Hierarchical Clustering AHC (Ward’s technique) with Euclidean distance measure. The calculus was performed using a set of individuals composed of 22 individuals (representing different collection sites and different months of collection) and the whole 11 fatty acids identified.

3 Results and discussion

3.1 Lipids extract

The lipid contents (range variation) obtained from air-dried Pistacia atlantica leaves harvested during several months are shown in Table 1 (the data are presented statistically). Dewaxed lipids content mean value increased gradually throughout leaf development, (Fig. 1); it seems that the maximum mean value was reached in November at the last stage of development (3.05% against 0.89% for full developing stage of leaves). In order to confirm the lipids yield evolution throughout this season, a statistical analysis involving ANOVA followed by Tukey’s tests were performed. In other words, the question was: is there significant difference between the mean yields for the four months? Indeed, results showed significant differences (95% of confidence at least) between the yields of May, July and November. The yield in September was not significantly different to both August and November. This close similarity could be explained by the fact that the September month is an intermediate state where the full developed phenological stage spread from August until the beginning of November. In this case, the lipids yields increased rapidly at the beginning of the phenological state May (young leaves), then increased very slowly to reach a steady state (a plateau) from the full until end of the developing phenological state (from August to November). Similar results were reported earlier, in which the total lipid content in Catharanthus roseus leaf, increased during expansion at full maturity. And with no marked changes occurred during aging [34].

Table 1
Composition of the major fatty acids and lipid contents obtained from P. atlantica leaves

Fatty acids May August September November

Min. Max. Mean sd Min. Max. Mean sd Min. Max. Mean sd Min. Max. Mean sd

C12:0 1.23 2.82 2.16 0.78 0.00 3.38 1.95 1.55 0.93 3.18 2.08 0.90 0.00 4.85 2.24 1.58

C14:0 2.94 4.79 3.82 0.76 4.30 8.76 6.53 1.60 6.80 15.42 9.94 3.50 10.78 18.55 13.81 3.17

C16:0 16.43 20.25 18.18 1.76 11.88 26.66 20.81 5.24 17.16 29.14 22.02 4.52 20.06 32.27 24.78 5.22

C16:1ω7 1.68 20.39 8.16 8.36 2.86 13.22 6.37 3.76 1.26 12.23 5.31 4.78 2.68 10.16 6.35 2.81

C17:0 1.03 2.68 1.66 0.75 0.00 10.43 3.02 3.72 0.89 2.62 1.75 0.60 0.00 4.23 2.37 1.44

C18:0 2.98 5.61 4.08 1.12 2.45 8.49 5.13 2.03 2.66 4.80 3.83 0.93 3.14 7.00 4.63 1.57

C18:1ω9 8.75 18.49 12.36 4.47 19.28 37.60 28.83 6.50 19.90 43.41 27.79 8.63 12.38 19.73 15.32 2.66

C18:2ω6 13.11 21.88 17.68 3.80 6.38 10.96 8.69 1.67 8.68 14.61 10.81 2.29 7.48 16.92 12.96 3.37

C18:3ω3 22.83 35.49 27.25 5.92 9.06 19.13 12.53 3.75 5.07 23.18 11.90 6.85 8.00 17.88 14.78 3.49

C20:0 1.05 2.64 1.93 0.71 0.00 7.42 3.24 2.48 0.94 3.59 2.21 0.92 0.00 5.25 2.12 1.79

C20:1ω9 1.37 6.33 2.73 2.40 0.00 7.60 2.92 2.89 1.31 4.29 2.38 1.10 0.00 1.93 0.66 0.85

SFA 26.44 36.53 31.82a 4.19 32.72 46.56 40.66ab 5.53 35.29 47.18 41.81ab 4.62 41.70 62.09 49.94b 7.95

MUFA 16.19 31.09 23.25a 6.68 32.20 45.61 38.12b 5.08 25.01 47.23 35.48b 7.78 17.34 31.38 22.33a 5.13

PUFA 35.94 57.37 44.93a 8.99 17.23 25.51 21.22b 2.84 15.97 32.09 22.71b 6.62 15.48 34.80 27.73b 6.61

UFA 63.47 73.56 68.18a 4.19 53.44 67.28 59.34ab 5.53 55.82 64.71 58.19ab 4.62 37.91 58.30 50.06b 7.95

UFA/SFA (%) 1.74 2.78 2.19 0.44 1.15 2.06 1.50 0.36 1.12 1.83 1.42 0.28 0.61 1.40 1.04 0.32

Lipid content (%) dw 0,64 1,51 0.89a 0.26 1.74 3.05 2.33b 0.43 1.70 3.24 2.42bc 0.62 2.38 3.60 3.00c 0.37

Fatty acids	May	August	September	November
C12:0	1.23	2.82	2.16	0.78	0.00	3.38	1.95	1.55	0.93	3.18	2.08	0.90	0.00	4.85	2.24	1.58
C14:0	2.94	4.79	3.82	0.76	4.30	8.76	6.53	1.60	6.80	15.42	9.94	3.50	10.78	18.55	13.81	3.17
C16:0	16.43	20.25	18.18	1.76	11.88	26.66	20.81	5.24	17.16	29.14	22.02	4.52	20.06	32.27	24.78	5.22
C16:1ω7	1.68	20.39	8.16	8.36	2.86	13.22	6.37	3.76	1.26	12.23	5.31	4.78	2.68	10.16	6.35	2.81
C17:0	1.03	2.68	1.66	0.75	0.00	10.43	3.02	3.72	0.89	2.62	1.75	0.60	0.00	4.23	2.37	1.44
C18:0	2.98	5.61	4.08	1.12	2.45	8.49	5.13	2.03	2.66	4.80	3.83	0.93	3.14	7.00	4.63	1.57
C18:1ω9	8.75	18.49	12.36	4.47	19.28	37.60	28.83	6.50	19.90	43.41	27.79	8.63	12.38	19.73	15.32	2.66
C18:2ω6	13.11	21.88	17.68	3.80	6.38	10.96	8.69	1.67	8.68	14.61	10.81	2.29	7.48	16.92	12.96	3.37
C18:3ω3	22.83	35.49	27.25	5.92	9.06	19.13	12.53	3.75	5.07	23.18	11.90	6.85	8.00	17.88	14.78	3.49
C20:0	1.05	2.64	1.93	0.71	0.00	7.42	3.24	2.48	0.94	3.59	2.21	0.92	0.00	5.25	2.12	1.79
C20:1ω9	1.37	6.33	2.73	2.40	0.00	7.60	2.92	2.89	1.31	4.29	2.38	1.10	0.00	1.93	0.66	0.85
SFA	26.44	36.53	31.82a	4.19	32.72	46.56	40.66ab	5.53	35.29	47.18	41.81ab	4.62	41.70	62.09	49.94b	7.95
MUFA	16.19	31.09	23.25a	6.68	32.20	45.61	38.12b	5.08	25.01	47.23	35.48b	7.78	17.34	31.38	22.33a	5.13
PUFA	35.94	57.37	44.93a	8.99	17.23	25.51	21.22b	2.84	15.97	32.09	22.71b	6.62	15.48	34.80	27.73b	6.61
UFA	63.47	73.56	68.18a	4.19	53.44	67.28	59.34ab	5.53	55.82	64.71	58.19ab	4.62	37.91	58.30	50.06b	7.95
UFA/SFA (%)	1.74	2.78	2.19	0.44	1.15	2.06	1.50	0.36	1.12	1.83	1.42	0.28	0.61	1.40	1.04	0.32
Lipid content (%) dw	0,64	1,51	0.89a	0.26	1.74	3.05	2.33b	0.43	1.70	3.24	2.42bc	0.62	2.38	3.60	3.00c	0.37

SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; UFA: unsaturated fatty acids; UFA/SFA: ratio “unsaturated/saturated” fatty acids; Mean: mean value relative to six different replicates (populations); sd: standard deviation. ^*UFA/SFA was not directly calculated by the ratio of values listed in the each column; rather that, this ratio was obtained by determining at a first step the ratio for each sample, in a second step, the tabulated ratio was determined using determined ratios in first step. Mean values followed by the same small letter within the same line are not significantly different (p < 0.05) according to Tukey’s (Multiple Range Test).

Fig. 1

Variation in lipids content (mean value of each month) during development of Pistacia atlantica leaves. Vertical bars indicate standard deviation error (±). Mean values followed by the same small letter over experimental points are not significantly different (p > 0.05) according to Tukey’s (Multiple Range Test).

The low lipid content recorded at first stage of development can be attributed to the growing process of the young leaves, while at the last phenological state the biosynthetic activity of the lipids is at its highest rate. This final stage where leaf color is tending to yellow is considered a critical state since shortly afterwards leaf began to fall (aging and senescence). The gradual increase pattern may be explained by the fact that lipid content in Pistacia atlantica leaves might be regulated with the intervention of the enzymatic system of fatty acid synthetase (FA-Synthetase) which operates differently during this last stage. In other words, FA-Synthetase process was induced in some manner which resulted in higher production rate of lipids (accumulation). In the opposite way, at first stage, enzymes were qualified by a phenomenon called retro-inhibition in which they were more or less inactive. Hence, lipid production was at its lowest content. Accordingly, and in some recent works, it was also reported that the biosynthesis of lipids in fruits does not initiate at early stage of the development [35 –37].

Changing weather conditions might also provide an explanation for the seasonal variation in the lipid content. Actually, lipids biosynthesis is known as sensitive to temperature. This parameter plays a crucial role in the composition of the oil FA by affecting the process regulating of FA-desaturase enzyme. As a consequence, in this study, lower temperatures at full and at last developing stages may be partially responsible of the higher lipids contents and especially in November where temperature reaches it lower value. This hypothesis was validated by Larkindale, et al. [38] who suggested that the lipid biosynthesis increases considerably at lower temperature.

The lipids accumulation may also be affected by other factors than temperature, such as light intensity exposure and daylight length, which are dependent on the season. It seems that decreasing day light and time exposure from May to November are both factors responsible for increasing lipid content through this growing period, in which lipid biosynthesis were more favorable.

3.2 Fatty acids composition

The results of gas chromatography GC and gas chromatography coupled to the mass spectroscopy GC/MS analysis of the fatty acid methyl esters (FAME) of P. atlantica leaves obtained during different harvest months are shown in Table 1. In all samples, the range of main saturated fatty acids (SFA) detected were palmitic acid (C16:0 = 11.88–32.27%) followed by myristic acid (C14:0 = 2.94–18.55%), margaric acid (C17:0 = 0.0–10.43%) and stearic acid (C18:0 = 2.45–8.49%). On the other hand, the main unsaturated fatty acids (UFA) were oleic acid (C18:1 = 8.75–43.41%) followed by α-linolenic acid (C18:3 = 5.07–35.49%), linoleic acid (C18:2 = 6.38–21.48%), palmitoleic acid (C16:1 = 1.26–20.39%) and gondoic acid (C20:1 = 0.0–7.6%), although the others were found to be of low overall abundance.

For the overall samples, UFA fractions compositions showed the highest percentages ranging from 37.91 to 73.56% which were mostly represented by high amounts of poly unsaturated fatty acids (PUFA) in the range of (15.48–57.37%). Alternatively, the SFA were varying from 26.44 to 62.09%.

The results of ANOVA combined with Tukey’s tests showed significant difference between the contents of SFA in May and November. Same result was obtained with UFA. This means that there is a significant difference between the beginning and the end of the developing state for the leaves. This result is quite in agreement with the case of lipids yield, at the same extent. Ones again, the intermediate months were not significantly different, nevertheless, the mean variations values of SFA and UFA were evolving progressively: increasing for SFA and simultaneously decreasing for UFA (Fig. 2). Similar report finding was in accordance with obtained evolution paces for both SFA and UFA [34, 39]. Indeed, with the progress of leaf expansion, the saturated fatty acids (capric, lauric, myristic, palmitic and stearic) increased while unsaturated fatty acids (oleic, linoleic and stearic) were found to decrease when changes in lipid profile during growth and senescence of Catharanthus roseus leaf was investigated by Mishra, et al. [34].

Fig. 2

Bars representation of the variation in unsaturated (UFA) and saturated (SFA) fatty acids compositions of the Pistacia atlantica leaves during development (mean values were considered). Vertical bars indicate standard deviation error (±). Mean values followed by the same letter over similar colored bars are not significantly different (p > 0.05) according to Tukey’s (Multiple Range Test).

For the first stage of leaf development (May), the average percentage of UFA was higher than SFA, and the difference was the highest one during the season, but after progressive evolution throughout the months until the last stage of development (November), the SFA and UFA percentages were converting to exactly same values (Table 1 & Fig. 2).

The identification of the FAME showed some clear variability of the compositions of the leaves during development. The mean percentage of May (first stage) was significantly different to the rest of the months (Fig. 3). Same results of previous analysis were confirmed for α-linolenic and linoleic acids. When mean values were considered, the total PUFA and its main components such as α-linolenic and linoleic acids showed the same accumulation gait ranging from 44.93±8.99% to 21.22±2.84% and from 27.25±5.92% to 12.53±3.75%, and from 17.68±3.80 to 8.69±1.67%, respectively, which decreased significantly from the first to the middle development stage (from May to August). Then it increased slightly towards the last development stage (November), or at least, remains practically steady until the end of the phenological state taking in consideration the non significant difference (p < 0.05) between the months of: August, September and November. It should be noted that PUFA are essential to human development and health. Moreover, they are highly important in cancer risk reduction, and also required to improve cardiovascular illnesses [40] and, due to their role in preventing inflammation and cerebro-vascular diseases, they are also studied as potential therapeutic agents for neuro-protective effects [41].

Fig. 3

Bars representation of the variation in poly unsaturated fatty acids (PUFA), C18:3 and C18:2 compositions of the Pistacia atlantica leaves during development (mean values were considered). Vertical bars indicate standard deviation error (±). Mean values followed by the same letter over similar colored bars are not significantly different (p > 0.05) according to Tukey’s (Multiple Range Test).

It should be highlighted that α-linolenic acid was the dominant UFA at the beginning of leaf formation in May where it presented a maximum mean of 27.25±5.92%, then, its amounts approached closely those of linoleic acid for the remaining development stages. It is worth noting that α-linolenic acid shows many protective effects (cardiovascular-protective, anti-cancer, neuro-protective, anti-osteoporotic, anti-inflammatory, and antioxidative effects) against a myriad of diseases [42]. Oleic acid was the main MUFA in all samples. It presented contrasting accumulation pace compared to α-linolenic and linoleic acids (Fig. 4). It increases starting from May to reach its maximum value for a long middle development stage period (37.60±28.83%, 43.41±27.79% in August and September, respectively); after that its decrease was once again towards its practical initial value at last development stage when leaves were of autumn colour. This analysis was confirmed by the Tukey’s test which has confirmed the presence of two similar pairs of different groups: May/November and August/September.

Fig. 4

Bars representation of the variation in saturated fatty acids (SFA) and poly unsaturated fatty acids (PUFA) compositions of the Pistacia atlantica leaves during development (mean values were considered). Vertical bars indicate standard deviation error (±).

According to previous studies, the oxidative stability of P. atlantica fruit oil was associated with high content of oleic acid [43]. Inversely, for this study, leaves lipids were associated with higher content of linolenic acid and especially in May. The hight contents of both linoleic and linolenic acids at the beginning of the season might be attributed to the increase in the activity of desaturase enzyme at this specific period. In a previous work which dealt with leaves part, it was demonstrated that the linolenic acid content of the leaves extracts decreased over heat stress [38]. This was in agreement with the results of this report in which the linolenic acid content decrease significantly in summer period (August) and then remained significantly steady in September and even in November which is not expected and where temperature decreased at the end of the phonological state. The alterations (deterioration of membranes) that occur within cells of senescing leaves at last phonological state might be responsible of the low contents of both linoleic and linolenic acids. Alterations in cell membrane composition and integrity reflect changes in membrane permeability. In addition, and similar to this last report, the loss of linolenic acid was correlated with significant increases of palmitic acid. In the present study, an increase in lipid saturation of leaves occurred during investigated months. This is primarily due to a decrease in linolenic acid and an increase in linoleic and palmitic acids. According to Larkindale, et al. [38] similar saturation increases have been observed in many plants. For a nutritional point a view comparison of main FA contents of investigated plant with a well known food rich nutrition plant date palm (Phoenix dactylifera L.) shows that investigated oil was more interesting. In fact, seed oil date palm was rich in oleic acid but its of content of linoleic and especially linolenic acid was very low [44].

Regarding the SFA, for the overall samples, palmitic acid showed its highest mean values at the last stage of leaf development (24.78±5.33%), followed by myristic acid detected with higher percentage (13.81±3.17%) at the same period. Indeed, for both components, it seemed that the mean accumulation increased gradually during leaf development. This observation was not confirmed by Tukey’s analysis for palmitic acid, since no significant differences was found between the means of the months. In this situation, the mean values of palmitic acid could be considered steady along the season.

At last, for the following components: C17:0, C18:0, C20:0 and C20:1, it seems that their mean values were practically steady over the season (Fig. 4).

We had noted with interest that the significant proportion of α-linolenic acid found in the leaves within first development stage (May) was very higher than those reported in previous published studies, thus, Samani, et al. [45], reported 1.09% for Atlas pistachio seeds from Iran. Trabelsi, et al. [46], registered 13.18% and 0.68% at the first and last stages of maturation for P. lentiscus fruit from Tunisia. Belyagoubi-Benhammou, et al. [47], obtained 0.44% for P. lentiscus fruit from Algeria. In fact, the obtained percentages of α-linolenic acid found in the leaves within first development stage (May) were similar to those of Chelghoum, et al. [28]. But, due to a reduced number of samples for this last report, no significant change of the whole reported FA and their different classes was noticed for the consecutive studied months of July, September and October. Similarly, in this study and except for the month of May, the remaining months showed no significance in the change of some specific FA components such as: C18:0, C18:2&3 and also for PUFA fraction.

The UFA/SFA ratio of unsaturated fatty acids to saturated fatty acids was not steady, it decreased from the maximum 2.19±0.44% observed in May to reach 1.04±0.32% in the last development stage. These values would supply to the oil beneficial properties in the first stage of development. As confirmed in this study, many reports which have analyzed either the entire lipid extract of photo-synthetic tissue or that of the chloroplast alone have shown a decline of more than 50% in the unsaturated/saturated esterified fatty acid ratio during senescence [34].

Finally, it should be noted that C12:0 was not reported existing in the leaf lipids by Chelghoum, et al. [28]. At the opposite side, both C20:0 and C20:1 are not identified in the current investigation, and which were reported by Chelghoum, et al. [28]. Except for C18:3, highest values for the following major components C18:0, 1, 2 were recorded in comparison with the previous study of Chelghoum, et al. [28].

3.3 Total tocopherols content

In recent years it has become evident that the different forms of tocopherols (vitamin E isomers) have antioxidant properties. They play an important role in cancer disease prevention and health promotion Li, et al. [48]. Hence, the study of tocopherols in P. atlantica leaves is of a major interest. The results of total tocopherol content (TTC) range variations in the dewaxed lipid fractions of P. atlantica leaves are shown in Table 2. The range variation was satrting from 1.55 to 4.26 mg/g of lipids. This range was pretty similar to the one determined recently for leaves of P. lentiscus tree from Algeria (3.2–4.6 mg/g of lipids) [49], but notably higher than those recently published (0.21–1.53 mg/g of lipids) for the same part and the same tree [28]. This higher content might be explained by the fact that when the crude lipids were dewaxed, the tocopherol compounds were obtained with higher quantities. Nevertheless, the obtained range was higher than those obtained by Benalia, et al. [50] for the pumpkin Cucurbita pepo L. seeds oil (0.10–0.22 mg/g of lipids) and by Guenane, et al. [51] who also registered 0.34 and 0.51 mg/g of oil for mature and immature P. atlantica fruits, respectively. Furthermore it is very important to mention that the results were higher than those of fruit oil found in other plants of the anacardiaceae family such as P. terebinthus var. Chia growing wild in Turkey as reported by Matthäus, et al. [52] and which recorded only 0.39 and 0.51 mg/g of oil.

Table 2
Total tocopherols (TTC) and total carotenoids (TCC) contents in P. atlantica leaves during development expressed as mg (α-tocopherol/β-carotene) equivalents/g lipids. Analysis of the differences between the categories performed with a confidence interval of 95%

Contents (mg/g lipids) May August September November

Min. Max. Mean sd Min. Max. Mean sd Min. Max. Mean sd Min. Max. Mean sd

TTC 1.55 3.41 2.28a 0.84 2.35 3.79 2.88ab 0.56 2.39 4.26 3.26b 0.64 2.39 3.67 3.00ab 0.41

TCC 0.20 0.60 0.13a 0.18 0.19 0.39 0.26ab 0.07 0.16 0.37 0.26ab 0.09 0.10 0.25 0.16b 0.05

Contents (mg/g lipids)	May	August	September	November
TTC	1.55	3.41	2.28a	0.84	2.35	3.79	2.88ab	0.56	2.39	4.26	3.26b	0.64	2.39	3.67	3.00ab	0.41
TCC	0.20	0.60	0.13a	0.18	0.19	0.39	0.26ab	0.07	0.16	0.37	0.26ab	0.09	0.10	0.25	0.16b	0.05

Mean: mean value relative to six different replicates (populations); sd: standard deviation. Mean values followed by the same small letter within the same line are not significantly different (p < 0.05) according to Tukey’s (Multiple Range Test).

As consequence, P. atlantica leaves seem to be rich in TTC. So, in order to evaluate the differences in TTC, the main previous factor was examined, which is harvest month (leaf development).

The obtained results showed significant differences in TTC according to the leaf development. It appears that the maximum of TTC is practically produced in September, towards last development stage, the TTC seemed to decrease. This observation was consolidated by Tukey’s test which showed significant difference between mean values for May and September (Table 2). Comparison of our results with other published data presented by Gourine, et al. [22] in which they reported that the seasonal variation showed that most of the main components of the essential oils reached their maximum values in September.

According to literature [53], tocopherols (Toc) play diversified roles in plant growth and physiological processes. Irrespective of form, Toc plays vital roles in development, signal transduction, interacting, or controlling phytohormonal regulation, senescence, and so on. The forms and levels of Toc are altered depending on plant growth stage and those perform different metabolic functions. It is also recognized that Toc plays a role in plants’ nutrient translocation, accumulation, and vascular systems and in carbohydrate translocation in various plant parts through phloem. Tocopherol is an antioxidant and thus has a clear role in plants’ tolerance to abiotic stress.

3.4 Total carotenoids content

It has been recognized that a diet rich in carotenoids, brought by the regular consumption of fruits and vegetables suggests a protective effect against cancer and cardiovascular diseases [54, 55]. These compounds play an important role in human vision because of their antioxidant properties and can filter blue light from the eye macula [56, 57]. Tocopherols and carotenoids improve oil stability and thus oils naturally rich in these constituents are preferred [58, 59]. The results of total carotenoid contents (TCC) in the dewaxed lipid fractions obtained from the leaves of P. atlantica are summarized in Table 2. The whole range variation was from 0.1 to 0.6 mg/g of lipids, which were presenting important variations. Contrarily to TTC case, significant difference between first and last month (beginning and end of development stages) were determined. For middle development (August and September), TCC were not significantly different (Table 2). TCC mean value increased from the beginning stage (May) to reach a plateau (steady state) during full developing stage, and then decreased once again to reach a mean value very close but slightly higher than obtained at the beginning stage. Comparison with recent published work for both same tree part showed that obtained TCC range was clearly higher than reported range of 0.0186–0.0866 mg/g lipids [28]. Once again, this difference could be attributed to the pre-treatment of crude oil by the dewaxing step.

3.5 Antioxidant activity

To estimate the antioxidant capacity, two antioxidant assay methods were investigated which are DPPH and β-carotene bleaching methods. To our knowledge, no studies have been done on the impacts of the potential influences of the main factor (harvest month) on the total antioxidant activities of lipid fraction of P. atlantica leaves during development. Table 3 summarizes the results for the antioxidant properties (EC₅₀ values) of P. atlantica leaves for both assays. The comparison of the different results showed that the antioxidant activities in all samples, at different regions over development stages had varied in the same way for both assays (DPPH and β-carotene bleaching methods).

Table 3
Variation of antioxidant activity of lipid fractions of P. atlantica leaves using DPPH and β-carotene bleaching assays

Antioxidant activity (mg/mL)

Months of collection May August September November

Min. Max. Mean sd Min. Max. Mean sd Min. Max. Mean sd Min. Max. Mean sd

DPPH assay EC₅₀ 7.76 13.17 10.32a 2.46 3.91 7.72 5.88b 1.51 5.04 7.33 6.25b 0.80 3.48 7.65 6.01b 1.58

β-carotene assay AA₅₀ 8.18 12.92 10.55a 1.90 3.99 8.56 6.98b 1.62 5.63 8.67 7.28b 1.20 5.63 9.70 7.74b 1.58

Antioxidants of reference DPPH assay EC₅₀ (μg/mL)±sd β-carotene assayAA₅₀ (μg/mL)±sd

Vitamin E 4.22±0.02 7.68±0.61

BHA 6.91±0.07 —

	Antioxidant activity (mg/mL)
DPPH assay EC₅₀	7.76	13.17	10.32a	2.46	3.91	7.72	5.88b	1.51	5.04	7.33	6.25b	0.80	3.48	7.65	6.01b	1.58
β-carotene assay AA₅₀	8.18	12.92	10.55a	1.90	3.99	8.56	6.98b	1.62	5.63	8.67	7.28b	1.20	5.63	9.70	7.74b	1.58
Antioxidants of reference	DPPH assay EC₅₀ (μg/mL)±sd	β-carotene assayAA₅₀ (μg/mL)±sd
Vitamin E	4.22±0.02	7.68±0.61
BHA	6.91±0.07	—

sd: standard deviation. Mean values followed by the same small letter within the same line are not significantly different (p < 0.05) according to Tukey’s Multiple Range Test.

First, the Tukey’s test applied for both antioxidant assays (DPPH and β-carotene) showed significant difference between first month of May and the rest of the months. Explicitly, August, September and November were not significantly different (Table 3).

Regarding DPPH results, the values of EC₅₀ found for the different samples during leaf development stages varied between 3.48 and 13.17 mg/mL. DPPH results were not far different from those obtained by Gourine, et al. [22] which reported EC₅₀ values of leaf essential oils for P. atlantica ranging from 8.80–27.48 mg/mL. The antioxidant activities were significantly higher than those presented by Guenane, et al. [51] who obtained EC₅₀ values for P. atlantica fruit oil, ranging from 17.1 to 51.3 mg/mL. In the same context, it useful to remind that, ethanolic extracts of Pistacia lentiscus berries growing in Algeria were investigated recently and showed strong in vitro antioxidant activities using DPPH, ABTS and reducing power assays [60]. Comparison with obtained DPPH activities with those of activities of a several varieties cultivars of date palm (Phoenix dactylifera L.) seed oils shows that investigated oil was by far more active than reported ones (46.42–77.58 mg/mL) [44].

According to different stages of leaf development, the decrease of EC₅₀ mean values was significant from the beginning until the middle stage of leaf development (August); subsequently, the values of EC₅₀ remained very close and steady (no significant difference). It is important to remind that the lowest antioxidant capacity is reflected by the highest EC₅₀ value. In this case, the antioxidant activity in May was lower than the rest of the months. Lipid extracts were less active compared to selected synthetic antioxidants (vitamin E, BHA) which showed very strong anti DPPH activities (Table 3). Comparison of obtained DPPH results with the previous study which dealt with the same plant part is practically impossible since different concentrations of initial DPPH solutions were employed [28].

β-carotene bleaching assay was applied for assessing antioxidant power of P. atlantica lipids fraction. The bleaching of β-carotene is a reaction caused by free radicals resulting from the hydroperoxides formed from linoleic acid. The rate of β-carotene bleaching can be decelerate in the presence of antioxidants by neutralization of linoleate free radical and other free radicals resulted through the system [61]. The AA₅₀ whole range variation values obtained with influence of the studied factor was ranged from 3.99 to 12.92 mg/mL (Table 3), it was practically the same range obtained with DPPH assay. The results of β-carotene bleaching test were very close to the range found for P. lentiscus leaves (Anacardiaceae) essential oil from Tunisia that was reported by Aissi, et al. [62] and who obtained a range variying from 5.57 to 11.20 mg/mL. The mean AA₅₀ value of 11.11±1.07 mg/mL registered in May was in accordance with the range assessed by Bachrouch, et al. [63] who obtained a range values extending from 10 to 11 mg/mL for essential oils of P. lentiscus leaves collected at the flowering stage (May). The best capacity was recorded in August for the leaves (AA₅₀ value 3.99 mg/mL) and the lowest capacity was observed for the same tree in fewer months earlier in May (AA₅₀ value 12.92 mg/mL). According to AA₅₀ mean values, the leaves exhibited their best antioxidant activity in August (AA₅₀ mean value 6.98±1.62 mg/mL). But statistically, the lowest antioxidant activity was recorded for developed stages (from August to November) with no significant difference. Likewise to DPPH case study for different stages of leaf development, the decrease of EC₅₀ mean values was significant from the beginning until the middle stage of leaf development (August); subsequently, the values of EC₅₀ come very close and steady.

As for the lowest antioxidant capacity, the highest AA₅₀ value was recorded for in the first development stage (May): 10.55±1.90 mg/mL. The obtained results of antioxidant capacity were substantially lower than those determined by the vitamin E, which presents a high capacity to prevent the bleaching of β-carotene.

3.6 Cluster analysis

For better understanding of similarities and/or differences between the studied samples in terms of FA compositions (Possible presence of region effect and/or month of collection on the FA composition), cluster analysis was performed using Agglomerative Hierarchical Clustering (AHC). The results of AHC schemed in the dendrogram of Fig. 5, showed two clearly distinguished clusters “or groups” of samples (cluster I: left cluster) and (cluster II: right one composed of two subgroups SG(1) & SG(2)). The presence of these groups and subgroups consolidated the initial theory of existing dissimilarity of the studied samples upon the variability of the chemical composition of the studied samples. A close examination of the first group (I) reveals that it enclosed whole samples collected for the consecutive months of August & September (middle developing stage: full expanded leaves). Alternatively, the second group (II) agglomerated mainly the samples of May (late stage of beginning of the formation of the young leaves) with those of November (last stage of losing leaves from the trees: yellow leaves). Moreover, the first subgroup SG(1) separates the samples of November from those of May which belongs this time to the SG(2). For SG(1), exception was made for one sample which belongs to September. So, for this sample it may be the presence of some over process for its phenological stage at this particular case. In order to better clarify and quantify the differences between the determined clusters, we have determined the range variations, the mean values and their standard deviation for all groups and subgroups. These results are summarized in Table 4.

Fig. 5

Dendrogram obtained from a cluster analysis of 22 individuals (representing different regions and different months of collection) and 11 variables (fatty acids obtained from Algerian Pistacia atlantica leaves). Samples are clustered using Ward’s technique with a Euclidean distance measure. S1-4: samples collected in May, S5-10: samples collected in August, S11-16: samples collected in September, S17–22: are samples collected in November.

Table 4

Range variation and mean values of the different clusters of the studied individuals obtained by CAH classification for the composition of the major fatty acids obtained from P. atlantica leaves

Fatty acids	Group (I)				Group (II)
					Subgroup SG(1)				Subgroup SG(2)
	Min.	Max.	Mean	sd	Min.	Max.	Mean	sd	Min.	Max.	Mean	sd
C12:0	0.00	3.38	2.11	1.21	0.00	4.85	2.05	1.52	1.23	2.82	2.16	0.78
C14:0	4.3	15.42	8.10	3.27	9.61	18.55	13.21	3.30	2.94	4.79	3.82	0.76
C16:0	11.88	29.14	21.02	4.73	20.06	32.27	24.91	4.77	16.43	20.25	18.18	1.76
C16:1ω7	1.26	13.22	6.02	4.28	2.68	10.16	5.99	2.74	1.68	20.39	8.16	8.36
C17:0	0.00	10.43	2.52	2.71	0.00	4.23	2.15	1.43	1.03	2.68	1.66	0.75
C18:0	2.45	8.49	4.45	1.73	3.14	7.00	4.66	1.43	2.98	5.61	4.08	1.12
C18:1ω9	19.28	43.41	29.07	7.14	12.38	19.9	15.97	2.98	8.75	18.49	12.36	4.47
C18:2ω6	6.38	14.61	9.83	2.30	7.48	16.92	12.38	3.44	13.11	21.88	17.68	3.80
C18:3ω3	5.07	19.13	11.22	4.18	8.00	23.18	15.98	4.50	22.83	35.49	27.25	5.92
C20:0	0.00	7.42	2.89	1.87	0.00	5.25	1.95	1.69	1.05	2.64	1.93	0.71
C20:1ω9	0.00	7.6	2.77	2.16	0.00	1.93	0.75	0.82	1.37	6.33	2.73	2.40
SFA	32.72	47.18	41.09	5.10	41.70	62.09	48.93	7.73	26.44	36.53	31.82	4.19
MUFA	29.59	47.23	37.87	5.49	17.34	31.38	22.71	4.79	16.19	31.09	23.25	6.68
PUFA	15.97	27.23	21.04	3.93	15.48	34.80	28.36	6.26	35.94	57.37	44.93	8.99
UFA	52.82	67.28	58.91	5.10	37.91	58.30	51.07	7.73	63.47	73.56	68.18	4.19
UFA/SFA^*	1.12	2.06	1.47	0.32	0.61	1.40	1.08	0.31	1.74	2.78	2.19	0.44

^*UFA/SFA was not directly calculated by the ratio of values listed in the each column; rather that, this ratio was obtained by determining at a first step the ratio for each sample, in a second step, the tabulated ratio was determined using determined ratios in first step.

For the case study of main similarities between all clusters, the three clusters are practically presenting similar mean values in the composition of the following SFA: C12:0; C17:0; C18:0 and C20:0. Now, the differences or the dissimilarities between these clusters are discussed as follows. The first group was mainly differentiated with its high percentage of UFA C18:1ω9, with a mean and sd variation of 29.07±7.14% with comparison of SG(1) (15.97±2.98%) and SG(2) (12.36±4.47%). The SG(2), was characterized by relatively higher percentages of UFA C18:3ω3 (27.25±5.92% vs “15.98±4.50% and 11.22±4.18% ”) and C18:2ω6 (17.68±3.80% vs “12.38±3.44% and 9.83±2.30% ”) when compared with the rest of the subgroup 1 and group (I), respectively. This SG(2) was also characterized by lower mean percentage of SFA C14:0 (3.82% against 13.21% and 8.10%). Finally, the SG(1) is characterized by relatively high mean percentage of C14:0 (13.21%) and C16:0 (24.91%) (Table 4), but the lowest mean value for the minor UFA C20:1ω9 (0.75%).

Alternatively, if we consider the difference between the obtained clusters upon the different total chemical classes (SFA, MUFA, PUFA and USF/SFA) we can observe that the SG(2) is mainly characterized by the highest mean value of PUFA (44.93% vs “28.36% and 21.04% ”). For this SG, the UFA was again slightly higher than those of the rest of the clusters (68.16% vs “51.07% and 58.91% ”). At the opposite side, SG(1) is also characterized by lower mean percentage of SFA (31.82% vs 48.93% and 41.09%). Furthermore, the UFA/SFA for this SG was the highest one (2.19±0.44). For the first group (I), it was distinguished mainly by its highest content of MUFA (37.87%) and by its lowest content of PUFA (21.04%), when compared to the rest of the clusters. The SG(1) presented the highest obtained mean value of SFA (48.93%) and the lowest UFA/SFA value (1.08±0.31). Some similarities were detected for the two SG(1&2) referring to their practically similar mean contents of MUFA (22.71% and 23.25%).

4 Conclusion

The current study showed that the harvesting month has a significant effect on the present material, concerning the lipids yield, the composition of the fatty acids and total contents of tocopherols and carotenoids. The increasing in the lipid content during development of Pistacia atlantica leaves (from May to November) was clearly detected. The lipids were rich in different and very important UFA. The SFA increased during leaves development while UFA decreased rapidly from first month to reach a steady state. The highest antioxidant activities and the percentages of both C18:3 and C18:2 were obtained for the month of May at the first development stage of leaves (young leaves). This month is considered as an optimal period for harvesting the leaves, which provide the most interesting nutritional potential in the food industries or for medicinal purposes.

Footnotes

Acknowledgments

The authors also wish to acknowledge the DGRSDT (Direction Générale de la Recherche Scientifique et du Développement Technologique) of the MESRS (Ministère de l’Enseignement Supérieur et de la Recherche Scientifique) of Algeria for the financial support granted for our research laboratory.

Author contributions

M.I. Benguechoua collected the plant samples, performed experiments and optimization and drafted the manuscript. M. Benguechoua supervised experiments. N. Gourine corrected the draft manuscript, checked the results and discussion, and supervised the work. A.M.S. Silva provided analysis. M. Saidi and M. Yousfi designed the study, supervised the work.

Conflict of interest

All authors state that they have no conflict of interest to declare.

References

Den Hartigh

. Conjugated Linoleic Acid Effects on Cancer, Obesity, and Atherosclerosis: A Review of Pre-Clinical and Human Trials with Current Perspectives. Nutrients. 2019;11(2):370.

Lordan

, Zabetakis

. Invited review: The anti-inflammatory properties of dairy lipids. J Dairy Sci. 2017;100(6):4197–212.

Tena

, Lobo-Prieto

, Aparicio

, García-González

. Storage and preservation of fats and oils. In: Ferranti P, Berry EM, Anderson JR, editors. Encyclopedia of Food Security and Sustainability. Oxford: Elsevier; 2019. pp. 605-18.

Rauf

, Patel

, Uddin

, Siddiqui

, Ahmad

, Muhammad

, et al. Phytochemical, ethnomedicinal uses and pharmacological profile of genus Pistacia. Biomed Pharmacother. 2017;86:393–404.

Ben Ahmed

, Yousfi

, Viaene

, Dejaegher

, Demeyer

, Heyden

. Four Pistacia atlantica subspecies (atlantica, cabulica, kurdica and mutica): A review of their botany, ethnobotany, phytochemistry and pharmacology. J Ethnopharmacol. 2021;265:113329.

Shams-Eldin

, Abdur-Rahman

. Chapter 22 - Cold pressed pistachio (Pistacia vera) oil. In: Ramadan MF, editor. Cold Pressed Oils: Academic Press; 2020. pp. 267-72.

Reboredo-Rodríguez

, González-Barreiro

, Cancho-Grande

, Simal-Gándara

, Giampieri

, Forbes-Hernández

, et al. Effect of pistachio kernel extracts in MCF-7 breast cancer cells: Inhibition of cell proliferation, induction of ROS production, modulation of glycolysis and of mitochondrial respiration. J Funct Foods. 2018;45:155–64.

Catalán

, Alvarez-Ortí

, Pardo-Giménez

, Gómez

, Rabadán

, Pardo

. Pistachio oil: A review on its chemical composition, extraction systems, and uses. Eur J Lipid Sci Technol. 2017;119(5):1600126.

Monjauze

. Connaissance du bétoum Pistacia atlantica Desf. Revue Forestière Française. 1980;32(4):356–63.

10.

Yousfi

, Nedjemi

, Belal

, Ben Bertal

. Étude des acides gras de huile de fruit de pistachier de l’Atlas algérien. Oléagineux, Corps gras, Lipides. 2003;10(5-6):425–7.

11.

Benhassaini

, Bendahmane

, Benchalgo

. The chemical composition of fruits of Pistacia atlantica Desf. subsp. atlantica from Algeria. Chem Nat Compd. 2007;43(2):121–4.

12.

Bentireche

, Guenane

, Yousfi

. Fatty acids, the unsaponifiablematter, and polyphenols as criteria to distinguish Pistacia atlantica unripe fruit oil. J Am Oil Chem Soc. 2019.

13.

Guenane

, Bombarda

, OuldElhadj

, Yousfi

. Effect of Maturation Degree on Composition of Fatty Acids and Tocopherols of Fruit Oil from Pistacia atlantica Growing Wild in Algeria. Nat Prod Commun. 2015;10(10):1723–8.

14.

Bahmani

, Saki

, Asadbeygi

, Adineh

, Saberianpour

, Rafieian-Kopaei

, et al. The effects of nutritional and medicinal mastic herb (Pistacia atlantica). J Chem Pharm Res. 2015;7(1):646–53.

15.

Yousfi

, Djeridane

, Bombarda

, Duhem

, Gaydou

. Isolation and characterization of a new hispolone derivative from antioxidant extracts of Pistacia atlantica. Phytother Res : An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives. 2009;23(9):1237–42.

16.

Achili

, Amrani

, Bensouici

, Gül

, Altun

, Demirtas

, et al. Chemical constituents, antioxidant, anticholinesterase and antiproliferative effects of Algerian Pistacia atlantica Desf. extracts. Recent patents on food, nutrition & agriculture.. 2020;11(3):249–56.

17.

Benabderrahmane

, Aouissat

, Jordan Bueso

, Bouzidi

, Benali

. Chemical composition of essential oils from the oleoresin of Pistacia atlantica Desf. from Algeria. J Biochem Int. 2015;2(4):133–7.

18.

Falahati

, Sepahvand

, Mahmoudvand

, Baharvand

, Jabbarnia

, Ghojoghi

, et al. Evaluation of the antifungal activities of various extracts from Pistacia atlantica Desf. Curr Med Mycol. 2015;1(3):25–32.

19.

Benabdallah

, Kouamé

, El Bentchikou

, Zellagui

, Gherraf

. Études ethnobotanique, phytochimique et valorisation de l’activité antimicrobienne des feuilles et de l’oléorésine du pistachier de l’atlas (Pistacia atlantica Desf.). Phytothérapie. 2015;15(4):222–9.

20.

El Zerey-Belaskri

, Cavaleiro

, Romane

, Benhassaini

, Salgueiro

. Intraspecific chemical variability of Pistacia atlantica Desf. subsp. atlantica essential oil from Northwest Algeria. J Essent Oil Res. 2017;29(1):32–41.

21.

Gourine

, Yousfi

, Bombarda

, Nadjemi

, Stocker

, Gaydou

. Antioxidant activities and chemical composition of essential oil of Pistacia atlantica from Algeria. Ind Crops Prod. 2010;31(2):203–8.

22.

Gourine

, Yousfi

, Bombarda

, Nadjemi

, Gaydou

. Seasonal Variation of Chemical Composition and Antioxidant Activity of Essential Oil from Pistacia atlantica Desf. Leaves. J Am Oil Chem Soc. 2010;87(2):157–66.

23.

Nadhir

, Ibrahim

, Emile

, Mohamed

. Chemical composition of the essential oil of unripe galls of Pistacia atlantica Desf. from Algeria. The Nat Prod J. 2011;1(2):125–7.

24.

Gourine

, Yousfi

, Bombarda

, Nadjemi

, Stocker

, Gaydou

. Antioxidant activities and chemical composition of essential oil of Pistacia atlantica from Algeria. Ind Crops Prod. 2010;31(2):203–8.

25.

Benhammou

, Bekkara

, Panovska

. Antioxidant and antimicrobial activities of the Pistacia lentiscus and Pistacia atlantica extracts. Afr J Pharm Pharmacol. 2008;2(2):22–8.

26.

Sifi

, Dzoyem

, Quinten

, Yousfi

, McGaw

, Eloff

. Antimycobacterial, antioxidant and cytotoxic activities of essential oil of gall of Pistacia atlantica Desf. From Algeria. Afr J Tradit Complement Altern Med. 2015;12(3):150–5.

27.

Ben Ahmed

, Yousfi

, Viaene

, Dejaegher

, Demeyer

, Mangelings

, et al. Seasonal, gender and regional variations in total phenolic, flavonoid, and condensed tannins contents and in antioxidant properties from Pistacia atlantica ssp. leaves. Pharm Biol. 2017;55(1):1185–94.

28.

Chelghoum

, Guenane

, Harrat

, Yousfi

. Total Tocopherols, carotenoids, and fatty acids content variation of Pistacia atlantica from different organs’ crude oils and their antioxidant activity during development stages. Chem Biodivers. 2020;17(9):e2000117.

29.

Shahidi

, Ambigaipalan

, Wanasundara

PKJPD

. Extraction and Analysis of Lipids. In: Akoh CC, editor. FOOD LIPIDS: Chemistry, Nutrition, and Biotechnology. Fourth ed: CRC Press, Taylor & Francis Group; 2017. pp. 131-66.

30.

Emmerie

, Engel

. Colorimetric determination of α-tocopherol (vitamin E). Recueil des Travaux Chimiques des Pays-Bas. 1938;57(12):1351–5.

31.

Rutz

, Voss

, Zambiazi

. Influence of the Degree of Maturation on the Bioactive Compounds in Blackberry (Rubus spp.) cv. Tupy. Food Nutr Sci. 2012;3(10):1453.

32.

Molyneux

. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J Sci Technol. 2004;26(2):211–9.

33.

Sun

, Ho

C-T

. Antioxidant activities of buckwheat extracts. Food Chem. 2005;90(4):743–9.

34.

Mishra

, Tyagi

, Singh

, Sangwan

. Changes in lipid profile during growth and senescence of Catharanthus roseus leaf. Braz J Plant Physiol. 2006;18:447–54.

35.

Glew

, Ayaz

, Millson

, Huang

, Chuang

, Sanz

, et al. Changes in sugars, acids and fatty acids in naturally parthenocarpic date plum persimmon (Diospyros lotus L.) fruit during maturation and ripening. Eur Food Res Technol. 2005;221(1):113–8.

36.

Schulz

, Borges

GdSC

, Gonzaga

, Seraglio

SKT

, Olivo

, Azevedo

, et al. Chemical composition, bioactive compounds and antioxidant capacity of juçara fruit (Euterpe edulis Martius) during ripening. Food Res Int. 2015;77:125–31.

37.

Tlili

, Mejri

, Yahia

, Saadaoui

, Rejeb

, Khaldi

, et al. Phytochemicals and antioxidant activities of Rhus tripartitum (Ucria) fruits depending on locality and different stages of maturity. Food Chem. 2014;160:98–103.

38.

Larkindale

, Huang

. Changes of lipid composition and saturation level in leaves and roots for heat-stressed and heat-acclimated creeping bentgrass (Agrostis stolonifera). Environ Exp Bot. 2004;51(1):57–67.

39.

El Qarnifa

, El Antari

, Hafidi

. Effect of maturity and environmental conditions on chemical composition of olive oils of introduced cultivars in Morocco. J Food Qual. 2019;2019:1854539.

40.

Chen

, McClements

, Decker

. Design of foods with bioactive lipids for improved health. Annu Rev Food Sci Technol. 2013;4:35–56.

41.

Walters

, Hackett

, Caesar

, Isaacson

, Mosconi

. Role of nutrition to promote healthy brain aging and reduce risk of Alzheimer’s disease. Curr Nutr Rep. 2017;6(2):63–71.

42.

Kim

K-B

, Nam

, Kim

, Hayes

, Lee

B-M

. α-Linolenic acid: Nutraceutical, pharmacological and toxicological evaluation. Food Chem Toxicol. 2014;70:163–78.

43.

Jamshidi

, Hejazi

, Golmakani

M-T

, Tanideh

, Heidari Esfahani

. Preventive effect of different wild Pistachio oils on oxidative stress markers, liver enzymes, and histopathological findings in a metabolic syndrome model. Galen Med J. 2019;8:e1238.

44.

Laghouiter

, Benalia

, Gourine

, Djeridane

, Bombarda

, Yousfi

. Chemical characterization and in vitro antioxidant capacity of nine Algerian date palm cultivars (Phoenix dactylifera L.) seed oil. Med J Nutrition Metab. 2018;11:103–17.

45.

Samani

, Zareiforoush

, Lorigooini

, Ghobadian

, Rostami

, Fayyazi

. Ultrasonic-assisted production of biodiesel from Pistacia atlantica Desf. oil. Fuel. 2016;168:22–6.

46.

Trabelsi

, Cherif

, Sakouhi

, Villeneuve

, Renaud

, Barouh

, et al. Total lipid content, fatty acids and 4-desmethylsterols accumulation in developing fruit of Pistacia lentiscus L. growing wild in Tunisia. Food Chem. 2012;131(2):434–40.

47.

Belyagoubi-Benhammou

, Belyagoubi

, El Zerey-Belaskri

, Zitouni

, Ghembaza

, Benhassaini

, et al. Fatty acid composition and antioxidant activity of Pistacia lentiscus L. fruit fatty oil from Algeria. J Food Meas Charact. 2018;12(2):1408–12.

48.

G-X

, Lee

M-J

, Liu

, Yang

, Lin

, Shih

, et al. δ-tocopherol is more active than α-or γ-tocopherol in inhibiting lung tumorigenesis in vivo. Cancer Prev Res. 2011;4(3):404–13.

49.

Harrat

, Benalia

, Gourine

, Yousfi

. Variability of chemical composition of fatty acids, tocopherols and the antioxidant activity of the lipids from the leaves of Pistacia lentiscus L. from Algeria. Med J Nutrition Metab. 2018;11:199–215.

50.

Benalia

, Djeridane

, Gourine

, Nia

, Ajandouz

, Yousfi

. Fatty acid profile, tocopherols content and antioxidant activity of algerian pumpkin seeds oil (Cucurbita pepo L). Med J Nutrition Metab. 2015;8(1):9–25.

51.

Guenane

, Bentireche

, Bellakhdar

, Ould Elhadj

, Yousfi

. Total tocopherol content and antioxidant activity of fruit oil from Pistacia atlantica Desf. growing wild in Algeria. Der Pharma Chem. 2017;9(13):153–7.

52.

Matthäus

, Özcan

. Quantitation of Fatty Acids, Sterols, and Tocopherols in Turpentine (Pistacia terebinthus Chia) Growing Wild in Turkey. J Agric Food Chem. 2006;54(20):7667–71.

53.

Hasanuzzaman

, Nahar

, Fujita

. Chapter 12 - Role of Tocopherol (Vitamin E) in Plants: Abiotic Stress Tolerance and Beyond. In: Ahmad P, Rasool S, editors. Emerging Technologies and Management of Crop Stress Tolerance. San Diego: Academic Press; 2014. pp. 267-89.

54.

Machlin

. Critical assessment of the epidemiological data concerning the impact of antioxidant nutrients on cancer and cardiovascular disease. Crit Rev Food Sci Nutr. 1995;35(1-2):41–9.

55.

Wright

, Mayne

, Swanson

, Sinha

, Alavanja

. Dietary carotenoids, vegetables, and lung cancer risk in women: the Missouri Women’s Healty Study (United States). Cancer Causes Control. 2003;14(1):85–96.

56.

Demmig-Adams

, Adams

. Eye nutrition in context: mechanisms, implementation, and future directions. Nutrients. 2013;5(7):2483–501.

57.

Bernstein

, Li

, Vachali

, Gorusupudi

, Shyam

, Henriksen

, et al. Lutein, zeaxanthin, and meso-zeaxanthin: the basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease. Prog Retin Eye Res. 2016;50:34–66.

58.

Warner

, Frankel

. Effects of β-carotene on light stability of Soybean oil. J Am Oil Chem Soc. 1987;64(2):213–8.

59.

Frankel

. Recent advances in lipid oxidation. J Sci Food Agric. 1991;54(4):495–511.

60.

Belhachat

, Aid

, Mekimene

, Belhachat

. Phytochemical screening and in vitro antioxidant activity of Pistacia lentiscus berries ethanolic extract growing in Algeria. Med J Nutrition Metab. 2017;10:273–85.

61.

Tang

, Shen

, Xie

, Liu

, Du

, Lin

,et al.. Physicochemical characterization, antioxidant activity of polysaccharides from Mesona chinensis Benth and their protective effect on injured NCTC-cells induced by H₂O₂. Carbohydr Polym. 2017;175:538–46.

62.

Aissi

, Boussaid

, Messaoud

. Essential oil composition in natural populations of Pistacia lentiscus L. from Tunisia: Effect of ecological factors and incidence on antioxidant and antiacetylcholinesterase activities. Ind Crops Prod. 2016;91:56–65.

63.

Bachrouch

, Msaada

, Wannes

, Talou

, Ksouri

, Salem

,et al.. Variations in composition and antioxidant activity of Tunisian Pistacia lentiscus L. leaf essential oil. Plant Biosyst. 2015;149(1):38–47.

	Antioxidant activity (mg/mL)
Months of collection	May				August				September				November
	Min.	Max.	Mean	sd	Min.	Max.	Mean	sd	Min.	Max.	Mean	sd	Min.	Max.	Mean	sd
DPPH assay EC₅₀	7.76	13.17	10.32a	2.46	3.91	7.72	5.88b	1.51	5.04	7.33	6.25b	0.80	3.48	7.65	6.01b	1.58
β-carotene assay AA₅₀	8.18	12.92	10.55a	1.90	3.99	8.56	6.98b	1.62	5.63	8.67	7.28b	1.20	5.63	9.70	7.74b	1.58
Antioxidants of reference	DPPH assay EC₅₀ (μg/mL)±sd								β-carotene assayAA₅₀ (μg/mL)±sd
Vitamin E	4.22±0.02								7.68±0.61
BHA	6.91±0.07								—