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Medicinal Plants From Lebanon: Effects of Essential Oils From Pistacia Palaestina on Proliferation and Erythroid Differentiation of Human Leukemic K562 Cells

Posted on: Sunday, 20 November 2005, 03:02 CST

By Lampronti, I; Saab, A; Gambari, R

Aim. Medicinal plants are used in prenatal care, in obstetrics, in gynecology, in respiratory disorders, in skin diseases, in cardiac pathologies, in nervous and muscular disorders, in mental health. In the present paper, we show that essential oil from Pistacia palaestina is able to inhibit the in vitro proliferation of human leukemic K562 cells.

Methods. After producing the essential oils from Pistacia palaestina, the in vitro antiproliferative activity and the induction of erythroid differentiation were assayed.

Results. These results suggest that the antiproliferative activity of Pistacia palaestina is not associated to induction of differentiation. However, when extracts from Pistacia palaestina are added to K562 cells together with suboptimal concentrations of mithramycin, a clear synergic effect was observed.

Conclusion. The results obtained demonstrate a novel antitumor property of Pistacia palaestina extracts, in addition to the previously reported anti-inflammatory, anti-ulcerative and hepatoprotective activities.

KEY WORDS: Medicinal plants * Antiproliferative activity * Erythroid differentiation * Essential oils.

Several medicinal plants have been described to exhibit a variety of therapeutic properties.1-4 For instance, medicinal plants are used in prenatal care,5,6 in obstetrics,7 in gynecology,6 in respiratory diseases,3,4 in skin disorders,8 in cardiac pathologies,4 in nervous and muscular disorders,2 in mental health.1 Examples are a variety of herbal drugs lowering cholesterol (useful in chronic angina and congenital heart diseases),'. 10 herbal treatments for a wide range of skin conditions,8 including diseases like psoriasis and erysipelas.1,2 Therefore, medicinal plants should be considered of great interest, since they could provide health security to rural people in primary health care. According to WHO estimates, more than 80% of people in developing countries depend on traditional medicines for their primary health needs.1"4

In the Mediterranean area, several medicinal plants have been described from Lebanon.11^s For instance, Barbour et al.1! tested the in vitro antimicrobial efficacy of extracts derived from different parts of 27 indigenous wild plant species that have been commonly used in Lebanese folk medicine. Nine test microorganisms were used, namely Escherichia coli, Proteus sp., Pseudomonas aeruginosa, Shigella dysenteria, Salmonella enteritidis, Salmonella typhi, Staphylococcus aureus, Streptococcus faecalis, and Candida albicans. One water extract from whole plant of Alchemilla diademata showed high antimicrobial activity against Staphylococcus aureus.

Figure 1.-A, B) Plant and seeds of Pistaciapalaestina. C) Kinetics of proliferation of K562 cells cultured for the indicated length of time in the absence ([black circle]), or in the presence of 0.1 ([black square]), 1 ([black triangle up]), 10 ([white circle]), 50 ([white square]) and 100 ([white triangle up]) g/mL of essential oil isolated from Pistacia palaestina.

However, it should be clearly stated that for most of medicinal plants from Lebanon very few biological data are available. 13

Among the most studied medicinal plants, Pistacia palaestina deserves attention.14-17 Pistacia palaestina Boiss. (Pistacia terebinthus L. var. palaestina) is a medicinal and foodstuff plant.18 The ripe fruits are used largely in the Middle East as a component of the so-called Zaatar, a mix of aromatic and food plants. Results of gas chromatography (GC) and gas chromatography/ mass spectroscopy (GC/MS) analyses of the essential oils of ripe and unripe fruits of Pistacia palaestina demonstrate that the oil is rich in monoterpenes, while only small amounts of sesquiterpenes have been detected in leaves, fruits and galls.19

The in vitro antimicrobial activity of essential oils from Pistacia against bacteria and fungi has been reported, together with the chemical composition obtained by GC/MS.20-22 In addition, Pistacia has been shown to exert anti-ulcerative,23 anti- inflammatory 24 and hepatoprotective 25 activities.

In this communication, we determined the biological activity of essential oil preparations from Pistacia palaestina against a human tumor cell line.

In fact, it should be underlined that medicinal plants can give rise to antitumor extracts.26-2? For instance, in a recent paper antitumor activity of Emblica officinalis has been reported.28 Aqueous extracts of Emblica officinalis were found to display cytotoxic effects on L929 cells in culture in a dose-dependent manner. Interestingly, Emblica officinalis extracts were found to reduce ascites and solid tumors in tumor-bearing mice.29-30

In order to determine whether Pistacia palaestina exhibits antiproliferative activity, we analyzed the effects of essential oil from this plant on the human chronic myelogenous leukemia K562 cell line.31'33 In addition, the ability to induce erythroid differentiation was determined.

Materials and methods

Essential oils from Pistacia palaestina

The essential oils from Pistacia palaestina were produced as follows. The seeds of Pistacia palaestina were collected in El- mathen Province, Republic of Lebanon. The plant material seed is aromatic and has been known for many years to be a natural source for an essential oil, obtained by hydro-distillation. Fresh seeds of Pistacia palaestina were hydro-distillated in a distillation apparatus under high pressure, the oil recovered from the Florentine receivers was dried over a hydrous sodium sulphate and finally filtered through Whatman #1 paper.

Assays of in vitro antiproliferative activity

The in vitro antiproliferative activity of essential oils from Pistacia palaestina was assayed as follows. Cell number/ml was determined by using a model ZBI Coulter Counter (Coulter Electronics, Hialeah, FL, USA). Usually, cells were seeded at the initial cell concentration of 310^sup 4^ cells/mL and the cell number/mL determined after 2, 3, 4, 5 days of cell culture. IC^sub 50^ was determined usually after 4 days, when untreated cells are in the log phase of cell growth.

Assays of in vitro induction of erythroid differentiation

Erythroid differentiation was determined by counting benzidine positive cells after suspending the cells in a solution containing 0.2% benzidine in 0.5 M glacial acetic acid, 10% H^sub 2^O^sub 2^, as elsewhere described.31-33 Induction of differentiation was compared with that obtained using well established inducers of differentiation of K562 cells, such as cytosine arabisonside,34 mithramycin,35 angelicin,36 hydroxyurea37 and butyric acids.38 Synergism with mithramycin was studied by treating K562 cells with plant extracts in the presence of sub-optimal concentrations of mithramycin.

Results

The methanolic solutions of Pistacia palaestina were obtained as a liquid material starting from the corresponding hydro-distillated fractions, derived from seeds. The fractional distillation is a technique used to purify natural product. Several apparatus distillation systems are available, such as crude oil, natural product and essential oil distillation systems. Hydro-distillation is a process that allows the extraction of the essential molecule structure of medicinal plant, using a flask and a condenser apparatus. The methanolic solution of Pistacia palaestina is heated and the resulting vapor steam is captured, condensed and collected. The biological activities of Pistacia palaestina essential oils were compared to those of Emblica officinalis, previously reported to exhibit antitumor effects.28

Figure 2.-Effects of increasing concentrations of extracts from Pistacia palaestina ([black square]), Emblica officinalis ([black circle]) and Thuya orientalis ([white triangle up]) on cell growth of K562 cells. Cells were cultured for 4 days as indicated and the cell number/mL determined and compared to the value obtained using control untreated K562 cells.

Effects of Pistacia palaestina extracts on in vitro proliferation of human leukemic K562 cells

Figure 1 shows the effects of increasing amounts of the Pistacia palaestina essential oil on cell proliferation of K562 cells. K562 cells were seeded at the initial cell concentration of 30 000 cells/ mL and then cultured for the indicated length of time in the absence or in the presence of 0.1, 1, 10, 50, and 100 μg/mL of essential oil. As it can be easily noted, 50 μg/mL were able to inhibit cell growth of K562 cells, while 100 μg/mL fully suppress cell proliferation. In order to determine the IC50 (concentrations of extracts leading to 50% inhibition of K562 cell growth), we determined the values of cell number/mL after 4 days, when untreated cells are in the log phase of cell growth.28

Figure 2 shows the results of this experiment. In these assays, the essential oil of Pistacia palaestina (Figure 2, [black square]) was compared with extracts from Emblica officinalis (Figure 2, [black circle]) and essential oils from Thuya orientalis (Figure 2, [black triangle up]). The data obtained show that the IC^sub 50^ of Pistacia palaestina essential oil is about 50 g/mL (54.818 g/mL), a value that is higher than that obtained when Emblica officinalis extracts were used (0.4 g/ml), but significantly lower than the IC^sub 50^ values obtained with other medicinal plant extracts from Lebanon, such as Thuyaorientalis (Figure 2), Pinus pinea, Laurus nobilis leaves, Juniperus Oxycedrus seeds, Calamintha origanifolia, and Common fennel (data not shown).

Figure 3.-A) Effects of 0.5 g/mL of extracts from Pistacia palaestina ([white circle]) and 25 nM mithramycin ([black square]) on erythroid differentiation of K562 cells. B) Proportion of benzidine positive cells after 7 days culture as indicated.

Effects of Pistacia palaestina extracts on erythroid differentiation of human leukemic K562 cells

Several antitumor compounds are able to exert their antiproliferative activity by the activation of the terminal program of differentiation.32,35,39 Therefore, we tested the ability of the essential oil from Pistacia palaestina in inducing differentiation of K562 cells. Figure 3A shows that K562 cells do not undergo erythroid differentiation when cultured in the presence of 0.5 g/mL of extract. As control, we report that the DNA-binding compound mithramycin (25 nM) is, on the contrary, a potent inducer of erythroid differentiation of this tumor cell line. These results suggest that the antiproliferative activity of Pistacia palaestina (Figure 1C and Figure 2) is not associated to induction of differentiation.

However, when the essential oil from Pistacia palaestina is used in combination with sub-optimal concentrations of the inducing agent mithramycin, a clear synergic effect was obtained. This effect is reported in Figure 3B, which indicates that, while treatment with 25 nM mithramycin in associated with 879.5% of benzidine-positive cells, 5 nM mithramycin induces only erythroid differentiation in a very low proportion of cells (83.5%). Interestingly the combination of 5 nM mithramycin and 0.5 g/mL of Pistacia palaestina extracts leads to a significant increase in the proportion of benzidine- positive K562 cells, suggesting that, in these experimental conditions, induction of erythroid differentiation occurs. The extent of differentiation, despite being lower than that found with cytosine arabinoside and mithramycin, is similar to that stimulated by rapamycin, and higher to those induced by hydroxyurea and butyric acid (Table I).

Discussion and conclusions

A consistent proportion of people in developing countries depends on traditional medicines for their primary health needs.1-4 Accordingly, medicinal plants are used in prenatal care, in obstetrics, in gynecology, in respiratory disorders, in skin disorders, in cardiac diseases, in nervous and muscular disorders, in mental health.5-7

Despite the fact that several medicinal plants from Lebanon have been described, very few data on their biological activity are present in the literature.

From the data obtained, it is apparent that the essential oil from Pistacia palaestina was able to inhibit the in vitro proliferation of K562 cells.

TABLE I.-Level of K562 differentiation induced by known erythroid inducers and Pistacia palaestina, in synergic effect with sub- optimal concentration of mithramycin.

We like to underline that these data demonstrate a novel property of the essential oil from Pistacia palaestina extracts. Essential oil from Pistacia palaestina has been described to retain antimicrobial,40 antiulcerative,23 hypoglicemic24 and hepatoprotective25 activities.

For instance, Magiatis et al. studied the chemical composition of the three essential oils obtained by steam distillation of the mastic gum, leaves and twigs of Pistacia lentiscus by GC/MS and identified 69 constituents, reporting in the same paper the in vitro antimicrobial activity of the three essential oils and of the resin against 6 bacteria and 3 fungi.40 In addition, Pistacia lentiscus has been described for its anti-ulcerative properties; mastic, a concrete resinous exudate, obtained from the stem of this tree, at an oral dose of 500 mg/kg produced a significant reduction in the intensity of gastric mucosal damage in rats with experimentally- induced gastric and duodenal ulcers.23

Finally, the hepatoprotective effect of Pistacia lentiscus (aqueous extracts) was evaluated in vivo, utilizing carbon tetrachloride intoxicated rats as an experimental model. These studies demonstrated that bilirubin level and the activity of alkaline phosphatase (ALP) were both reduced upon treatment with P. latifolia, without reducing the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).25

As far as biological effects of P. palaestina reported in the present paper, we like to underlined that the antiproliferative activity of the studied essential oils is not associated to activation of the differentiation pattern of K562 cells. However, we found an interesting synergistic effect with mithramycin in inducing activation of erythroid differentiation (Figure 3B). In this respect, it is worth noting that a combined drug therapy in anticancer research has been proposed in several studies.41-43

TABLE II.-Bioactive molecules identified in Pistacia plant genus.

Extract from the essential oil from Pistacia palaestina should be therefore proposed for further investigations, in order to identify pure compounds suitable for antitumor differentiation therapy.

Possible candidates for the biological effects of medicinal preparation from Pistacia have been already identified, including α-pinene, myrcene, sabinene, limonene, (E)-ocimene, (Z)- ocimene, trans-verbenol, camphene, trans-pinocarveol (monoterpenes),44,21 isomasticadienonic acid, masticadienonic acid, 28-norolean-17-en-3-one (triterpenes).22 It is known that also sesquiterpenes and one linear nonterpenic compound are recognized in Pistacia plant family.45 A partial list of the identified bioactive molecules is shown in Table II.

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I. LAMPRONTI 1 A. SAAB 2, R. GAMBARI 1, 3

1 Biotechnology Center University ofFerrara, Ferrara, Italy

2 Department of Chemistry, Faculty of Sciences II University of Lebanon, Beirut, Lebanon

3 ER-GenTecb Department of Biochemistry and Molecular Biology University ofFerrara, Ferrara, Italy

R. Gambari is granted by AIRC, MIUR-COFIN (2002), MIUR-FIRB (2002), Fondazione Italiana Fibrosi Cistica, Associazione Veneta per Ia Lotta alia Talassemia (AVLT) and PRRIITT from Regione Emilia Romagna.

Address reprint requests to: Prof. R. Gambari, Dipartimento di Biochimica e Biologia Molecolare, Universit degli Studi di Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy. E-mail: gam@unife.it

Copyright Edizioni Minerva Medica Sep 2005

Source: Minerva Biotecnologica

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