*Article* **Insight into Analysis of Essential Oil from** *Anisosciadium lanatum* **Boiss.—Chemical Composition, Molecular Docking, and Mitigation of Hepg2 Cancer Cells through Apoptotic Markers**

**Hany Ezzat Khalil 1,2,\*, Hairul-Islam Mohamed Ibrahim 3,4, Hossam M. Darrag 5,6 and Katsuyoshi Matsunami <sup>7</sup>**


**Abstract:** Essential oils have been used in various traditional healing systems since ancient times worldwide, due to their diverse biological activities. Several studies have demonstrated their plethora of biological activities—including anti-cancer activity—in a number of cell lines. *Anisosciadium lanatum* Boiss. is a perennial aromatic herb. Traditionally, it is an edible safe herb with few studies exploring its importance. The current study aims to investigate the chemical composition of essential oil isolated from *Anisosciadium lanatum* using GC-MS, as well as report its anti-cancer potential and its mechanistic effect on HepG2 liver cancer cell lines, and conduct molecular docking studies. To achieve this, the essential oil was isolated using a Clevenger apparatus and analyzed using GC-MS. The cell viability of HepG2 liver cancer and normal fibroblast NIH-3T3 cell lines was assessed by MTT cytotoxicity assay. The effects of the essential oil on cell migration and invasion were assessed using wound healing and matrigel assays, respectively. The effect of the essential oil on migration and apoptotic-regulating mRNA and proteins was quantified using quantitative real-time PCR and Western blot techniques, respectively. Finally, computational docking tools were used to analyze in silico binding of major constituents from the essential oil against apoptotic and migration markers. A total of 38 components were identified and quantified. The essential oil demonstrated regulation of cell proliferation and cell viability in HepG2 liver cancer cells at a sub-lethal dose of 10 to 25 μg/mL, and expressed reductions of migration and invasion. The treatment with essential oil indicated mitigation of cancer activity by aborting the mRNA of pro-apoptotic markers such as BCL-2, CASPASE-3, CYP-1A1, and NFκB. The algorithm-based binding studies demonstrated that eucalyptol, nerol, camphor, and linalool have potent binding towards the anti-apoptotic protein BCL-2. On the other hand, camphor and eucalyptol showed potent binding towards the pro-apoptotic protein CASPASE-3. These findings highlight the effectiveness of the essential oil isolated from *Anisosciadium lanatum* to drive alleviation of HepG2 cancer cell progression by modulating apoptotic markers. Our findings suggest that *Anisosciadium lanatum* could be used as a phytotherapeutic anti-cancer agent, acting through the regulation of apoptotic markers. More well-designed in vivo trials are needed in order to verify the obtained results.

**Keywords:** *Anisosciadium lanatum*; GC-MS; HepG2; BCL-2; CASPASE-3; apoptotic markers

**Citation:** Khalil, H.E.; Ibrahim, H.-I.M.; Darrag, H.M.; Matsunami, K. Insight into Analysis of Essential Oil from *Anisosciadium Lanatum* Boiss.—Chemical Composition, Molecular Docking, and Mitigation of Hepg2 Cancer Cells through Apoptotic Markers. *Plants* **2022**, *11*, 66. https://doi.org/10.3390/ plants11010066

Academic Editors: Hazem Salaheldin Elshafie, Laura De Martino and Adriano Sofo

Received: 24 November 2021 Accepted: 23 December 2021 Published: 26 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Traditionally, medicinal and aromatic plants have been considered to play essential roles in the field of therapeutics all over the world [1]. Essential oils (Eos), as secondary metabolites produced from such medicinal and aromatic plants, offer great value in terms of their various curative and biological properties. Several investigations have demonstrated the anti-inflammatory, anti-oxidant, anti-fungal, anti-microbial, and cytotoxic activities of such Eos [2–4]. Cancer is described as a fatal health condition, considered to be one of major factors leading to death. Cancer affects all human beings and does not differentiate between gender or age, leading to severe negative health and socio-economic impacts. More than 75% of anti-cancer drugs are directly or indirectly derived from medicinal plants [5]. In this context, the discovery of new natural product candidates with anticancer properties has unique interest for the purpose of medical care [6]. Many cytotoxic molecules that are of plant origin are widely used in chemotherapy [7]. The contribution of food components to cancer assessment through lifestyle patterns has become popular in diet–disease investigations [8]. Food enriched with vitamins and bioactive phytochemicals could act as tumor-controlling agents to reduce cancer progression, especially in the case of liver or colon cancer [9].

Moreover, a few reports have revealed that Mediterranean EOs and diets based on substances such as vegetables, nuts, whole grains, olive oil, and/or fish oils can reduce cancer-related and total mortality rates [10,11]. Several reports have also investigated the antioxidant activities of essential oils and have shown them to be potent natural sources of antioxidants to control cancer. The abnormal cellular stress causes non-nuclear DNA damage, which leads to inhibition of protein transport and reactive oxygen species formation. The Apiaceae family (formerly Umbelliferae) is one of the families of flowering plants, which consists of 3780 species in 434 genera. It is distributed worldwide [12,13]. Investigations have proven that this family is rich in its diversity of phytochemicals that have been considered as potential sources of new therapeutic drugs, including terpenoids, saponins, flavonoids, coumarins, and poly-acetylenes. In addition, numerous species of the Apiaceae family are reputed to be a significant source of EOs. EOs isolated and identified from this family have been shown to contain more than 760 components in various chemical classes. The identified and reported constituents have shown substantial pharmaceutical and nutritional values [13–16]. Many members of the Apiaceae family possess various biological activities, such as anti-bacterial, hepatoprotective, cyclo-oxygenase inhibitory, and anti-tumor activities [12]. Most of the members are safe and edible plants. The *Anisosciadium* genus (Apiaceae family) comprises three species—*Anisosciadium isosciadium* Bornm., *Anisosciadium orientale* DC., and *Anisosciadium lanatum* Boiss.—and is endemic to Southwest Asia [17]. Previous investigations have reported on cytotoxicity and antioxidant assessments pertaining to isolated Eos from *Anisosciadium orientale* [17,18]. In this aspect, *Anisosciadium lanatum* Boiss (*A. lanatum*) is a member of the *Anisosciadium* genus that is native and spread wildly throughout the Arabian Peninsula, including Saudi Arabia [19,20]. *A. lanatum* is a perennial herb. Anatomically, its leaves are characterized by a bipinnately parted incision and clasping base. The inflorescences are of a compound umbel type. Its flowers are tiny and demonstrate whitish-pink petals. The bracts are spiny-tipped. The fruitlets of each secondary umbel are aggregated before ripening, but are later separated into spiny units [21,22]. Traditionally, Bedouins have used *A. lanatum* as local medicinal herb: A water extract of the dried aerial parts, including the stem, leaves, flowers, and fruits, is used for skin sores and boils [23]. The young green leaves are a refreshing palatable herb for Bedouin children [24,25]. Additionally, *A. lanatum* has demonstrated veterinary importance, being used in livestock treatment. The extract from the leaves and shoots has been used to treat skin conditions in goats and sheep [19]. Recent studies have reported that *A. lanatum* contains guaiane sesquiterpene and shows anti-proliferative activity towards liver, colon, and lung cells, as well as anti-mutagenic activities [25,26]. A simple study reported an analysis of the EO from *A. lanatum* [20]; however, no previous reports have detailed advanced analyses of EO isolated from of *A. lanatum* and its modulatory cytotoxic

effects, including those with respect to cell migration and invasion. In these contexts, the goal of the current exploration is to investigate *A. lanatum*, for the first time, with respect to its suggested potentials.
