Ethnobotanical investigation of Pistacia lentiscus L. grown in El Kala (Algeria), and phytochemical study and antioxidant activity of its essential oil and extracts

Abstract Ethnobotanical investigation was carried out using questionnaires among majority of El Kala (Algeria) inhabitants. The investigation found that lentisk (Pistacia lentiscus L.) is widely used in traditional therapy, especially in gastrointestinal and dermatological problems. Bioactive phytochemicals were determined by LC-MS/MS, and 14, 13, and 12 compounds were identified in leaves, stems and seeds, respectively. Flavonoids and phenolic acids were the most abundant compounds. Moreover, total phenols (306.5 mg GAE/g), flavonoids (95.25 mg RE/g), and condensed tannins (170.75 mg CE/g) contents were determined in leaves. Also, essential oil composition was investigated using GC/MS and 27 aroma compounds were identified. Monoterpene and sesquiterpene hydrocarbons were the most abundant. Besides, antioxidant activity was performed by DPPH, β-carotene bleaching, and ferrous ions chelating tests; and leaves extracts were more effective (IC50 of 2.75, 3.45, 13.5 µg/ml), than essential oils (IC50 of 10.5, 70.5, 157.25 µg/ml), and standards (trolox, 4.75; BHT, 5.25 µg/ml), respectively. Graphical Abstract


Introduction
Pistacia lentiscus L. is an evergreen shrub with a strong characteristic odour of the Anacardiaceae family (Pell 2004). It is widely distributed along Algeria's coastline and can grow even in arid areas (Charef et al. 2008). It has been used for a long time in folk medicine practice (Palevits and Yaniv 2000). In Turkey, the aerial part and/or resin, are used to treat ulcers, wounds, light burns, eczema, coughs, sore throats, asthma, kidney stones, stomachache, jaundice, paralysis, diarrhoea, and as antipyretic, anti-inflammatory, antibacterial, astringent, pectoral stimulant and antiviral (Duru et al. 2003). Its current use is limited to treat stomach aches, indigestion, jaundice, and respiratory disorders, according to independent ethnobotanical and ethnopharmacological surveys undertaken lately in Jordan and Palestine (Ali-Shtayeh et al. 2000;Lev and Amar 2002). In the treatment of tiny wounds, light burns, and erythema, traditional Algerian medicine mostly uses fatty oil obtained from mastic tree fruits. Orally, the oil is used to treat allergic respiratory issues and stomach ulcers. These uses are especially widespread in the East of Algeria, the oil is also widely used for the same indications in Tunisia (Grosjean 2011). P. lentiscus has several biological activities such as in vitro antioxidant (Aissi et al. 2016;Yosr et al. 2018), anti-Alzheimer (Aissi et al. 2016), and antimicrobial activities (Salhi et al. 2010;Mezni et al. 2015;Marengo et al. 2018).
Various parts of P. lentiscus contain medicinally important chemical compounds, including gallic acid, essential oil, anthocyanins and flavonol glycosides, resin, a-tocopherol, arabino-galactan proteins and nortriterpenoids (Nahida and Siddiqui 2012;Mezni et al. 2014). The chemical composition of P. lentiscus essential oil has several main compounds: a-pinene, b-caryophyllene, terpinen-4-ol, limonene, a-phellandrene, sabinene, a-pinene, p-cymene, c-cadinene, b-pinene, and c-terpinene (Negro et al. 2015;Leila et al. 2020). Despite ongoing researches, the aim of our study was to perform ethnobotanical investigation of P. lentiscus species through a survey about its traditional therapeutic uses to explore the potential of this plant in folk medicine. Besides, phytochemical investigations of both essential oil and methanol/water (80/20) extract and antioxidant activity were performed.

Ethnobotanical results
The obtained results from the ethnobotanical surveys showed that women used much more medicinal plants than men with percentages of 87.72% and 12.28%, respectively (Table S1). Several ethnobotanical investigations in Algeria (Miara et al. 2018;Beldi et al. 2021) have shown the dominance of women as main informant. Regarding the ages of participants, the results showed that the youth class carry more interest in medicinal plants compared to other age groups (Table S1). Illiterate people constitute the majority of medicinal plants users (35.09%), followed by those having a secondary level (22.82%) and finally, people with primary, middle and university level (14.03%) (Table S1). Such results were also observed by several authors in the south of Algeria (Miara et al. 2018;Beldi et al. 2021). The use of herbal remedies is more common among married people (85.5%) compared to a small percentage for single and divorced people. This can be explained by the responsibility of married people for their families. According to income, it turns out that the unemployed people recorded the highest rates of medicinal plants usage (71.93%) (Table S1). Other parameters concerning classification of users according to their sociographic profile are shown in Table S1.
From the 1176 ethnobotanical surveys were carried out in the study area, it was found that the most used plant by the local population was P. lentiscus with a rate of 9.69%, compared to the 111 other medicinal species (90.31%). The high UV value (1.46%) of P. lentiscus indicates its widespread use in traditional medicine in the studied region. Surveyed people showed that infusion is the most used method of preparation of P. lentiscus. The infusion allows to collect the most active substances and to reduce or eliminate the toxic effect of certain recipes (Salhi et al. 2010). Also, essential oil or powder is often used. Regarding the part used, majority of the surveyed people use the leaves and few of them use the fruits. The high frequency of the leaves usage could be explained by their abundance and availability all over the year or they are the storage site of the bioactive metabolites. The local Algerian people frequently use P. lentiscus for different diseases, mainly against digestive tract diseases and burns. Indeed, lentisk leaves are used for the treatment of dermatological diseases, stomachaches, pains, cough, bronchitis, eyes diseases, haemorrhoid, lung and liver disorders (Beldi et al. 2021). The high rate of use of this species for gastrointestinal ailments is explained by the nature of the diet in the studied area, like the excessive consumption of pasta, grilled meats, tobacco and alcohol, that causes digestive and circulatory diseases. Other reasons are the humid climate (respiratory diseases), and the rich vegetation and fauna (pollen grains, insect bites that cause allergies, asthma, eczema, etc.), which cause dermatological and other diseases, hereditary and genetic factors.

2.2.2.
Simultaneous determination of 37 phytochemicals in P. lentiscus extracts by LC-MS/MS 2.2.3. Total phenolic, flavonoid and tannin contents According to our data (Table S5), interesting amounts of total phenols (306.5 mg GAE/ g DW), flavonoids (95.25 mg RE/g DW), and condensed tannins (170.75 mg CATE/g DW) were obtained. Our results showed higher contents than those recorded from leaves, fruits, needles and flowers samples collected from males and female's species, at different phenological stages from Tunisia (Aissi et al. 2016).

Antioxidant activities:
Leaves extract was more effective (IC 50 of 2.75, 3.45, 13.5 mg/ml) than the tested standards, trolox (4.75 mg/ml) and BHT (5.25 mg/ml) for DPPH, Ferrous ion chelating and b-carotene bleaching activities, respectively. While, essential oil (IC 50 of 10.5, 70.5, 157.25 mg/ml) was less potent than leaf extract and the standards (Table S5). This could be explained by the high level of phenols in leaves, which revealed a more antioxidant potential. Similar results were reported by previous findings (Aissi et al. 2016). In addition, the antioxidant abilities of P. lentiscus essential oil are related to the high content of oxygenated monoterpenes and sesquiterpenes; the most efficient classes which provide redox potentialities by the neutralisation of radicals and the chelation of metal ions (Yosr et al. 2018).

Conclusion
The ethnobotanical survey revealed that women, young, illiterate and married people seem to be more attracted towards conventional medicine by using P. lentiscus which is administered in several ways, the most commonly one is infusion. People in northeastern Algeria eat a lot of pasta, which creates a lot of gastrointestinal problems, therefore they rely a lot on this plant to help them feel better. In the treatment of some diseases, the locals prefer to use leaves rather than seeds. In addition, the phytochemical study revealed that leaf EO is rich in monoterpenes and sesquiterpenes hydrocarbons, and the main component is a-pinene which characterises the leaf EO of P. lentiscus collected around Mediterranean countries. The leaf extract revealed a more antioxidant potential due to the high phenols content. Further and deeper investigations of in vitro and in vivo tests would be interesting to determine the proper dose for health benefits.