The ethanol fraction from the stem of Berberis libanotica inhibits the viability of adult T cell leukemia
IN C ER O V P A Y R M IG E H DI T C ® A
F. ESSEILY 1, M. EL EZZY 2, H. GALI-MUHTASIB 3, S. SAFI J. ESSEILY 4, M. DIAB-ASSAF 4, I. LAMPRONTI 5, A. SAAB
Aim. In the Mediterranean countries, several described medicinal plants are derived from Lebanon. According to Tohme et al. there are 2597 species in Lebanon. More than fifty two percent are endemic to Lebanon. In this paper we show that the ethanol fraction of the stem of Berberis libanotica is able to inhibit the viability of HTLV-1 positive (HuT-102) and HTLV-1 negative (CEM) cell lines of malignant T-cell leukemia. Methods. After traditional maceration to extract the ethanol fraction from Berberis libanotica stem, the in vitro viability was assayed. Results. The results suggest that Berberis libanotica (a Lebanese medicinal plant) contains a substantial amount of secondary metabolites such as alkaloids powerful in inhibiting the viability of HuT-102 and CEM cell lines. Conclusion. The obtained results demonstrate a novel anticancer property of Berberis libanotica stem extracts, in addition to the previously reported anti-inflammatory activity. Key words: Plants, medicinal - Antineoplastic agents - Ethanol - Leukemia-lymphoma, adult T-cell.
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edicinal plants are crucial for the treatment of several ailments and diseases. Decades ago the Indians utilized extracts from the roots of mayapple, Podophyllum peltatum, as an effective treatment for skin cancer. From this plant, researchers extracted the main constituent, podophyllotoxin, that was the lead compound of the anti-cancer agents called podophyllins, namely etoposide and teniposide.1 In the mediterranean areas, several medicinal plants described are from Lebanon. According to
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This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies (either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo, or other proprietary information of the Publisher.
MINERVA BIOTEC 2012;24:129-33
Corresponding author: Faculty of Public Health, section 2, Lebanese University, Campus Pierre Gemayel, sixth floor, Fanar. P.O.Box 90-1556 Jdeidet El Metn., Lebanon. E-mail:
[email protected]
Vol. 24 - No. 4
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1Laboratory
Science Department, Faculty of Public Health Lebanese University, Jdeidet El Metn., Lebanon 2Department of Biology, Faculty of Science 1 Lebanese University, Jdeidet El Metn., Lebanon 3 Department of Biology, American University of Beirut, Beirut, Lebanon 4Department of Biology, Faculty of Science 2 Lebanese University, Jdeidet El Metn., Lebanon 5Department of Biochemistry and Molecular Biology University of Ferrara, Ferrara, Italy
Tohmé et al.2 there are 2597 species in Lebanon. Several Lebanese medicinal plants were tested and showed to have significant antimicrobial effects. Extracts from 27 Lebanese plants such as Achillea damascena (whole plant) and Origanum libanotica showed significant antimicrobial activity against Staphylococcus aereus.3 In addition to the antimicrobial activity, essential oil from the Lebanese medicinal plants, such as Pistacia palaestina and Satureja montana, have shown antiproliferative and differentiation inducing effects in human erythroleukemic cells K562.4, 5 The ferutinin from extracts of many ferula genera particularly Ferula hermonis have shown to increase the release of calcium that induced DNA fragmentation through the activity of caspases 3 leading to apoptosis of Jurkat T cell line. Berberis libanotica belongs to the Berberidaceae family. It is an important Lebanese medicinal plant that is endemic to Lebanon. The vernacular name is Lebanon barberry found in regions such as Aarsal, Jabal, Barouk, Sir, Ehden, Qomoua. It is an endangered species due to the effect of grazing by the baladi goats that favor these kinds of plants in the sum-
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Materials and methods Collection of plant material Berberis libanotica was collected from Ehden, North governorate “mohaafazah”, Lebanon on March 17th 2010 at an altitude up to 1521 m and Botanical identity was authenticated by Prof. S. Safi (Biology Department, Faculty of science II, Lebanese University, El Fanar). A voucher sample was deposited in the Herbarium of Department of Biology, American University of Beirut. The plant stem was open air dried under the shade as previously reported. The plant stem was pulverized using a wooden pestle and mortar into moderately coarse powder. Then the powder was additionally ground by an electronic mill until a fine powder was obtained.
IN C ER O V P A Y R M IG E H DI T C ® A
mer.6 Berberis libanotica roots or commonly known as Shalesh El Barbary have an anti-inflammatory and neurotropic effect. In addition, its leaves are used for the treatment of arthritis and muscular pain. It is normally found at high altitudes, between 1200-1400 m and it is a shrubby tree of 15-50 cm. It has blackish or reddish woody branches, yellow spines, glabrous leaves 10-15 x 3-5 mm elliptical lanceolate and sessile yellow flowers whose petals and sepals (tepals) resemble each other 4-5 mm; its fruit is a blackish and oval berry. Blooming time is between May and June. The berry is edible but it causes digestive disorders in children. Adult T cell leukemia was identified as a distinct clinical entity in 1977 in Japan. ATL is a rare disease constituting about 2% from all hematological neoplasms. It is a highly aggressive form of leukemia/lymphoma characterized by highly malignant proliferation of CD4 T cell infected by the retrovirus HTLV-1. Studies showed that only 3-5% of infected individuals by HTLV-1 develop ATL after a very long latency period (40-60 years on average).7 The disease is characterized by its intrinsic resistance to conventional and high dose chemotherapy,8 aggressive clinical course, infiltration to numerous sites (gastrointestinal tract, skin and lung), hypercalcemia and the presence of leukemic cells having indented or multilobulated nuclei (flower like cell).9 A great deal of evidence supports the association between HTLV1 infection and ATL. After transmission and infection, HTLV-1 immortalizes the infected T cells in vivo, after which the virus establishes a long persistent infection. This immortalization of the infected T cell is either cytokine dependent or cytokine independent. This key event of transformation is not sufficient for the transformation of ATL since only a fraction is transformed. Further genetic and epigenetic changes and immune response weakening are the prerequisites for such a disease.10 The current standard therapy for ATL is combination therapy with AZT and IFNα.11, 12 Continuous therapy is required as relapse will occur if the treatment is stopped. Despite the significant survival advantage of the AZT/IFNα combination, most patients with acute ATL eventually relapse and die. Thus, despite the options available, additional novel targeted therapies are warranted for improved long-term survival in patients. In this study, we provide evidence that the ethanol extract from the stem of Berberis libanotica reduces the viability of both HTLV-1 positive cells (HuT-102) and HTLV-1 negative peripheral T-cell lymphomas (CEM).
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This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies (either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo, or other proprietary information of the Publisher.
THE ETHANOL FRACTION FROM THE STEM OF BERBERIS LIBANOTICA
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Preparation of Berberis libanotica ethanol extract
The utilized extraction method was reported by Gritsanapan.13 Briefly, traditional maceration was used since it yielded more crude extract better than percolation and soxhlet extraction. 10 g of powdered stem material was extracted using ethanol (99.7-100%) (C2H5OH-HPLC-HyperSolv) (provided by Chemical Laboratory of Faculty of Health II, Lebanese University). The extract was filtered using Whatman paper number 1 and concentrated to dryness using a rotator evaporator. Moreover, these crude extracts were freeze-dried using liquid nitrogen. Mass yield was calculated. For the mass yield from the extraction of the stem the values were 4.655% ethanol. The dried extract was stored at 4 °C. Cell lines
Two malignant T-cell leukemia cell lines (HUT102, CEM) were used. Both cell lines were kindly provided by Dr. Ali Bazarbachi, American University of Beirut. HUT-102 cells are ATL-derived HTLV-1 infected CD4+ T-cell lines that constitutively express the retrovirus HTLV-1. HuT-102 were originally derived from peripheral blood of a 26 year old black male with cutaneous T cell lymphoma with type C retrovirus particles. CEM are uninfected human T-cell line established from patients with T-cell leukemia. CEM cells are T-lymphoblastoid cell lines derived from peripheral blood of a Caucasian female with acute lymphoblastic leukemia (ALL) as described by Foley et al.14
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The HTLV1 positive and negative cells were grown in RPMI 1640 supplemented with 25 mM Hepes, 10% heat-inactivated Fetal Bovine Serum (GibcoBRL, Paisley, Scotland), 100 U/ml penicillin, and 100 g/mL streptomycin (Gibco-BRL, Paisley, Scotland) in a humidified incubator (37 °C) at 95% air, 5% CO2. For our experiments, cells were seeded on 35 mm culture plates, left to grow to 50% confluency, and then treated with defined concentrations of Berberis libonatica dissolved in ethanol. The control was treated with ethanol only. The ethanol concentration in control and treated cells did not exceed 0.1%.
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Results and discussion
Antineoplastic effects of ethanol extracts of Berberis libanotica effect of ethanol extract of libanotica on CEM
Berberis
Ethanol extracts from stem bark of Berberis libanotica inhibited the viability of CEM by 70.4% at 25 µg/mL and 82% at 50 µg/mL after 72 hours of treatment. The extent of inhibition in cell viability reached 92% at 100 µg/mL after 72 hours of treatment (Figure 1). Calculated IC50 was 5 μg/mL. effect of ethanol extract of libanotica on HuT-102
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Figure 1.—Ethanol extracts of stem bark of Berberis libanotica treatment induced a dose dependent inhibition of proliferation of CEM cell limes. Cells were treated at 50% confluency with either
