β-Caryophyllene, a Compound Isolated from the Biblical Balm of Gilead (Commiphora gileadensis), Is a Selective Apoptosis Inducer for Tumor Cell Lines

The biblical balm of Gilead (Commiphora gileadensis) was investigated in this study for anticancerous activity against tumor cell lines. The results obtained from ethanol-based extracts and from essential oils indicated that β-caryophyllene (trans-(1R,9S)-8-methylene-4,11,11-trimethylbicyclo[7.2.0]undec-4-ene) is a key component in essential oils extracted from the balm of Gilead. β-Caryophyllene can be found in spice blends, citrus flavors, soaps, detergents, creams, and lotions, as well as in a variety of food and beverage products, and it is known for its anti-inflammatory, local anaesthetic, and antifungal properties. It is also a potent cytotoxic compound over a wide range of cell lines. In the current paper, we found that Commiphora gileadensis stem extracts and essential oil have an antiproliferative proapoptotic effect against tumor cells and not against normal cells. β-caryophyllene caused a potent induction of apoptosis accompanied by DNA ladder and caspase-3 catalytic activity in tumor cell lines. In summary, we showed that C. gileadensis stems contain an apoptosis inducer that acts, in a selective manner, against tumor cell lines and not against normal cells.


Introduction
For a normal living cell to replicate accurately, it must go through several processes: precisely reproduce its DNA, produce sufficient cellular organelles, membranes, and so forth, to enable the survival of the daughter cells, and divide its DNA, cytoplasm, and organelles equally to form two functioning daughter cells [1][2][3]. These processes are part of a well-controlled equilibrium which exists between cell proliferation and cell death, two stages of the cell cycle that have become appreciated targets for intervention against cancer as the cancerous process results from an imbalance between the two [1]. This imbalance is linked with changes in the cell's response to signals from within. These signals include: self-sufficiency in growth signals, insensitivity to growthinhibitory (antigrowth) signals and limitless replicative potential (over proliferation), sustained angiogenesis, tissue invasion, metastasis, and evasion of apoptosis. Each of these physiologic changes occurs during tumor development [1,3,4].
Apoptosis, the programmed cell death, comprises two main phases: a commitment to cell death followed by an execution phase [5]. The apoptotic process is a cascade reaction, carried out and regulated by specialized cellular machinery, a family of cysteine proteases called caspases. The appearance of these caspases seems to be the biochemical event that defines a cellular response as apoptosis more than any other [6]. The caspase family is divided into two: the initiator 2 Evidence-Based Complementary and Alternative Medicine  caspases (caspase-8 and caspase-9) and the executioner caspases (caspase-3, -6, and -7) [7]. An apoptotic death stimulus (stimuli is plural) activates the initiator caspases which, in turn, activate the executioner caspases. The active executioners promote apoptosis by cleaving cellular substrates, which induces the morphological and biochemical features of apoptosis [8][9][10].
In modern cancer research, the isolation of active compounds from natural products for drug use has reached an all-time high in modern westernized medicine, resulting in most of the existing anticancer drugs [11,12]. These drugs are aimed specifically at various components of the intracellular signal transduction pathways controlling cell cycle, programmed cell death (apoptosis), or angiogenesis [9]. These naturally derived products are offering the world of medicine a great opportunity to evaluate new chemical classes of anticancer agents, as well as novel mechanisms of action [13].
In this study, we describe a new apoptosis-inducing agent, β-caryophyllene (trans-(1R,9S)-8-methylene-4,11,11trimethylbicyclo[7.2.0]undec-4-ene) which was found to be a major component in the essential oil derived from C. gileadensis. β-caryophyllene, a compound found in spice blends, citrus flavors, soaps, detergents, creams and lotions, and also in a variety of food and beverage products, [14,15] is known for its anti-inflammatory, local anaesthetic, and antifungal properties [16][17][18]. In experiments conducted on tumor cell lines, β-caryophyllene has been reported to have a potent cytotoxic activity over a wide range of cell lines [19,20].
Commiphora gileadensis (L.) (Syn C. opobalsamum, Amyris gileadensis, Amyris opobalsamum, Balsamodendron opobalsamum [21]) is a member of the highly studied and commercially used resinous plant family Burseraceae, comprising, among others, the biblical frankincense and myrrh [22]. It is native to southwest Arabia and Somaliland, where it grows in a thorn-bush formation under arid tropical conditions, often accompanied by shrubs and trees such as Balanites aegyptiaca and species of Maerua, Ziziphus [23]. It is a thornless shrub (1.5 m high) or small tree (5 m high) with a reddish or grayish bark which grows in hot deserts and semideserts. Its white flowers create small clusters and produce ovoid or ellipsoid, smooth, glabrous fruits which contain a fragrant yellow seed that brightens in color as the seed matures and dries [23][24][25] (Figures 1 and 2). After receiving a specimen sheet from his student Peter Forsskål in Yemen, Linnaeus gave it the name Commiphora gileadensis, in connection with the Bible's "balm of Gilead" (Genesis 37 : 25, Jeremiah 46 : 11, and Jeremiah 8 : 22). The specimen was sent with a letter dated June 3rd 1763, in which Forsskål wrote about his discovery of the "balm of Gilead" [21,25,26]. Ancient writers, such as Josephus Flavius, Pliny the elder, Pedanius Dioscorides, and Gaius Tacitus, highly praised it (the balm (balsam) tree of Judea) for its use as holy oil and in perfumes, but also considered it as a cure for many diseases [23,27]. The shoots of C. gileadensis are used in folk medicine to treat various illnesses [28][29][30][31][32][33]. However, the history and elaborate folk medicinal value of the species has not yet been put to the test and remains generally overlooked by modern science.
In this manuscript, we describe the antiproliferative and proapoptotic effect of the C. gileadensis-derived essential oil and its derivative compound β-caryophyllene.

Extraction. Commiphora gileadensis stem extracts were prepared as follows.
(1) Ethanolic stem extracts: stems were dried at 40 • C for three days and stem powder was suspended in tubes with ethanol 96% (EtOH-FRUTAROM) at a ratio of 200 μg/mL which were incubated overnight at room temperature (25 • C), followed by centrifugation (13,000 revolutions per minute (rpm)). The supernatant (the extract) was moved to another tube and kept at −20 • C until used.

EBV Transformation.
To establish the MoFir cells, B cells from whole blood were separated by Ficoll-Hypaque density gradient centrifugation. The cells were infected with the B95-8 strain of the Epstein-Barr virus. The RPMI medium (Biological Industries Beit Haemek) was used for cell culture. The EBV B lymphocyte cell line was maintained in RPMI, supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. The culture was maintained at 37 • C in 5% CO 2 atmosphere. The medium was changed every three days.
Three dilutions were examined in the experiment: 1 : 20000, 10000, and 5000. Stock solutions of Gilead oil dilutions were prepared by adding 1 μL to 20, 10, and 5 μL ethanol, respectively. 1 μL from the stock solution was added into the 1000 μL cell growth medium.

Results Presentation.
The results presented in this study are from a representative experiment. A minimum of five experiments were carried out with each compound, and the samples were analyzed in triplicates.
Cell death was assessed by the incubation of cells with Tetrazolium bromide salt MTT. The production of formazan was monitored using an ELISA reader at a wavelength of 450 nm. Cytotoxicity in the compound-treated culture was expressed as follows: % death = 100 × absorbance of compound-treated cell absorbance of ethanol-treated cells .
Apoptosis was induced by incubating the cell lines with the compounds in their normal serum-supplemented growth medium.  The caspase-3 cellular activity assay was carried out according to the manufacturer's instructions. For measuring the specific inhibition by Ac-DEVD-CHO, cell extracts were preincubated with the inhibitor (0.05 μM) for 10 minutes before the addition of the substrate. The DNA ladder analysis was performed as described before [37].

Results
We first investigated whether C. gileadensis extracts had antiproliferative effects against tumor cell lines using the MTT assay. We evaluated its effect on mouse (BS-24-1) and human (MoFir) cell lines using ethanol-(EtOH-) based stem extracts. As shown in Figure 3, following 24 h incubation of cells with extracts, cell survival was 30 and 50% for BS-24-1 cells and MoFir cells at 0.5 μL/well, respectively. IC 50 was 0.3125 and 2.5 μL/mL, respectively.
Based on the latter results, we assessed the effect of C. gileadensis essential oil on tumor cell lines, comparing it to two reference compounds: staurosporine (STS) and citral [35] (Figure 4). Proliferative inhibition of Gilead oil incubated with tumor cell lines for 2 h showed concentration dependency (Figure 4), and Gilead oil concentration of 1 : 5000 killed 87% of BS-24-1 cells and 40% of MoFir cells, suggesting that one or more of the components in the essential oil contributed to the tumor cell lines killing effect. We next separated and identified the Gilead oil volatiles components using GC-MS. The compound list received from the fractionation ( Table 1) was composed mainly of terpens from which we decided to focus on what was most likely to be the active compound, β-caryophyllene, known for its cytotoxicity, anti-inflammatory, and antifungal activity [16][17][18][19][20]38]. To examine the effect of β-caryophyllene, both  Figure 6). Pretreatment of Mofir and BS-24-1 cells with the specific caspase-3 inhibitor, Ac-DEVD-CHO, inhibited caspase-3 activity, indicating that the active enzyme in the assay in both cell lines is indeed caspase-3 (Figure 7). The property of β-caryophyllene as an inducer of caspase-3 enzymatic activity was compared and found similar to the reference compound citral (Figure 8).
A biochemical hallmark of apoptosis is the activation of endonucleases, leading to the fragmentation of the genomic DNA, which produces a characteristic ladder when separated on an agarose gel electrophoresis [39]. When C. gileadensis stem extracts were incubated with BS-24-1 for 24 h, a DNA ladder was observed (Figure 9). The incubation of C. gileadensis stem extracts with normal cells (FB) and MoFir for 24 h produced a DNA ladder only in MoFir (the tumor cell line, Figure 10). 24 h incubation of Gilead oil with BS-24-1 formed a similar pattern ( Figure 11) that was repeated following 2 h treatment of BS-24-1 cells with β-caryophyllene ( Figure 12). These results suggest that the C. gileadensis stem extract is an apoptosis inducer that acts in

Discussion
Commiphora gileadensis stem extracts and essential oil showed antiproliferative proapoptotic effects (exhibited via DNA "ladder" and caspase-3 activation) in tumor cell lines, while there was no apoptosis induction in normal cell lines (FB) (Figure 10). β-Caryophyllene, one of the compounds in  the essential oil, is responsible for these effects. Our results agree with previous studies [16][17][18][19][20]38] which indicated that β-caryophyllene shows antiproliferative activity against tumor cell lines. To the best of our knowledge, this is the first time that the function of an apoptosis-inducing agent was assigned to β-caryophyllene. One of the main problems with  compounds used for chemotherapy is that they are nonselective, namely, they kill normal cells as well as cancer cells [40]. Interestingly, the results in the current study indicated that β-caryophyllene acts in a selective manner against tumor cell lines and not against normal cells. Since β-caryophyllene is a natural product used by humans on a daily basis [14,15]