Pharmacognosy Research

: 2010  |  Volume : 2  |  Issue : 4  |  Page : 211--214

Cytotoxic essential oil from Annona sengalensis Pers. leaves

AL Ahmed1, S. E. M. Bassem2, YH Mohamed3, MW Gamila2,  
1 Department of Health Information Technology of Jeddah Community College, King Abdul-Aziz University, Al- Rehab, 80283 Jeddah, Kingdom of Saudi Arabia
2 Department Pharmacognosy, National Research Centre Dokki, 12622 Giza, Egypt
3 Department Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt

Correspondence Address:
A L Ahmed
Pharmacognosy Department, Faculty of Pharmacy, Minia University, 61519 Minia
Kingdom of Saudi Arabia


The cytotoxicity against brine shrimp of the essential oil obtained from the leaves of Annona senegalensis Pers. (Annonaceae) was studied. The confirmation of this toxicity has been done by using selected tumor cell lines (A549, HT29, MCF 7, RPMI, and U251). The results showed that the total oil and its fractions have showed mild to moderate cytotoxicity in brine shrimp lethality bioassay with LC50 = 27.3 μg/ml, and against some human tumor cell lines. The total oil and its fractions were analyzed by gas chromatography/mass spectroscopy (GC/MS). Seventy three compounds were identified.

How to cite this article:
Ahmed A L, Bassem S, Mohamed Y H, Gamila M W. Cytotoxic essential oil from Annona sengalensis Pers. leaves.Phcog Res 2010;2:211-214

How to cite this URL:
Ahmed A L, Bassem S, Mohamed Y H, Gamila M W. Cytotoxic essential oil from Annona sengalensis Pers. leaves. Phcog Res [serial online] 2010 [cited 2021 Jan 22 ];2:211-214
Available from:

Full Text


Annona senegalensis is found widely distributed in Africa, Latin America, and Europe. The plant possesses several folk medicinal uses. The root bark is used for intestinal troubles and the bark is chewed for stomach ache. The stem, root, and bark are used to treat diarrhoea and gastrointestinal trouble, [8] whereas the stem bark and leaves are used for the treatment of skin cancer and leukemia. [9],[10]

In this study, the essential oil of A. senegalensis var. Senegalensis was studied for its cytotoxicity as well as its chemical composition, where 19 mono and sesquiterpenoids were identified in the volatile oil of the leaves and fruits. [11] No reports have been found in the literature on the biological activity of A. senegalensis essential oil. On these bases, the volatile oil of A. senegalensis, cultivated in Egypt, was prepared analyzed by gas chromatography/mass spectroscopy (GC/MS) and was screened for possible anticancer activity.

 Materials and Methods

Plant material

The leaves of A. senegalensis Pers. were collected from El-Qanater garden, Qalubeia Province, Egypt, and identified by Agric. Eng. Badia Diwan, Herbarium of Orman Botanical Garden, Giza.

Essential oil preparation

Exactly 2 kg of fresh leaves were sliced in small pieces (2-3 cm 2 ), and hydro-distillation was performed according to Egyptian Pharmacopoeia 1984. The essential oil was obtained in 0.021% w/w.

Analysis of volatile oil

Thin layer chromatography (TLC): The analysis was carried out on silica gel 60 F 254 , precoated plates, layer thickness: 250 μm (E. Merck, Darmstadt, Germany) and developed in different systems where Benzene:EtOAc (86:14) gave the highest resolution. The spots were visualized by UV and P-anisaldehyde/H 2 SO 4 reagent.

Flash column chromatography: 400 mg of the volatile oil was chromatographed on a column (1.5 Χ 25 cm) using silica gel (5-40 μm) and benzene:EtOAc (86:14) as eluting solvent, where 50 fractions (5-10 ml each) were collected and combined into three pools as guided by TLC. The pools after evaporation yielded P-A (105 mg), P-B (45 mg), and P-C (40 mg). The fractionation of P-A was effected over silica gel column (5-40 μm), using benzene:EtOAc (86:14); 50 fractions (3-5 ml each) were collected and combined into six pools (PA1-PA6) as guided by TLC.

GC/MS: GC/MS analysis were carried out using a Hewlett Packard 5890A-5970 GC-MS series with mass selective detector, 9144 HP 5Ms (crosslinked 5% PHME siloxane) 30 m Χ 0.25 μm, film thickness HP-5, 50C for 2 min, then 0C/min to 270C, injecting temperature: 270C; carrier gas He 20 ml/min, injecting volume: 25 μl, MS: in the EI mode at 70 eV, det.: 2300 IMEM, scan range: 47-400.

Cytotoxicity bioassays

Brine shrimp: A solution of sea water was made by dissolving 32.5 g (a natural blend of salts and trace element for sea water fish [Sera Company, Aquaristik Gmbh, D5138 Henisberg, Germany]) in distilled water (1 l). ca. 1 mg of brine shrimp, Artemia salina (leach), eggs was taken in a hatching chamber (22 Χ 32 cm). The hatching chamber was kept under an inflorescent bulb for 48 h for the eggs to hatch into shrimp larvae (nauplii). Then 50 mg of tested extracts/fractions; or 1 mg of pure compounds, dissolved in 5 ml of solvent in which they were soluble and from this, 5, 50, and 500 μl of each solution was transferred to vials corresponding to 10, 100, and 1000 μg/ml, respectively. Each dosage was tested in triplicate. The test vials and one control containing 500 μl of solvent were allowed to evaporate to dryness under nitrogen. Ten larvae (nauplii) of A. salina were transferred into each vial and the volume made into 5 ml with sea salt solution (Dimethyl sulfoxide, DMSO) immediately after adding the nauplii, 24 h later, the number of surviving shrimp at each dosage was counted and recorded. LC 50 values were determined statistically. [12]

Human tumor cell cytotoxicity assay (HTCC)

This cytotoxicity assay was carried out at the Ohio State University Comprehensive Cancer Centre with the cooperation of Professor John Cassady. From growing stock cultures, cells were inoculated into 96-well tissue culture plates on day one (D1) at appropriate concentrations (1000-2000 cells depending on the cell line), then incubated for 24 h. Test compounds were then added on day two (D2) in five log dilutions beginning with the highest soluble concentration, (four wells for each concentration). Simultaneously, negative controls (no treatment) and positive controls (adriamycin, five log dilutions) are included then ED 50 (Dose of a drug that is pharmacologically effective for 50% of the population exposed to the drug) was as calculated. [4]


The steam volatile dark yellow oil isolated from the fresh leaves of A. senegalensis Pers., cultivated in El-Qanater garden, Egypt, showed mild to moderate cytotoxicity in brine shrimp lethality bioassay with LC 50 = 27.3 μg/ml, and against some human tumor cell lines (HTCL) cf. [Table 1]. Preliminary TLC analysis of the oil performed on pre-coated silica gel 60 F 254 , showed an imaginable condensed pattern, as detected with the UV or spraying with anisaledehyde sulfuric acid reagent. GC analysis of the volatile oil afforded 181 peaks [Figure 1], of which only 27 compounds were identifiable by GC/MS analysis, as aided by data library and confirmed by comparison with published MS spectra. [8] To concentrate the minor components, the oil was fractionated on NP silica gel, eluted with benzene:EtOAc (6:4), and the obtained fractions grouped into three pools (P-A least polar, P-B, and P-C most polar) as guided by TLC. At 50 ppm, P-A showed the highest cytotoxicity (93%), in the brine shrimp lethality bioassay, whereas P-B and P-C showed 21 and 40% cytotoxicity, respectively. Thus, P-A was selected for further fractionation, which resulted in six pools possessing variable lethality percentages on brine shrimp larvae at 50 ppm as follows: PA1, PA2, and PA5 gave 0%; PA3: 12.5%, PA4: 49%, and PA6: 45%, and on HTCL in vitro as clarified in [Table 1]. The six pools were further reanalyzed by GC/MS. The interpretation of the GC/MS chromatograms led to identification of 46 other compounds. The total 73 components identified in the volatile oil and d its fractions are grouped in [Table 2].{Table 1}{Table 2}{Figure 1}


The main component identified in the moderately cytotoxic fraction P-4 is caryophyllene oxide (64.5%), which when tested in pure form proved to be devoid of cytotoxicity on the five HTCL tested, with moderate cytotoxicity on brine shrimp (LD 50 36.8 ppm). As the bioactivity did not increase significantly in the fractions, it could be safely concluded that the mild to moderate cytotoxicity of this volatile oil is apparently due to a synergistic effect exerted by its particular combination of oxygenated and nonoxygenated monoterpenes, and sesquiterpenes as well as the other components.


The GC/MS analysis and the HTCL cytotoxicity screening were performed at the Chemistry department, and the Ohio Cancer Centre, respectively, of Ohio State University, via generous facility provided by Dr. John M. Cassady.


1Leboeuf M, Cavι A, Bhaumik PK, Mukherjee B, Mukherjee R. The Phytochemistry of the annonaceae. Phytochemistry 1982;21:2783-813.
2Cavθ A, Cortes D, Figader B, Hocquemiller R, Laprevote O, Laurens A, et al. Phytochemical potential of tropical plants. New York: Plenum Press; 1993. p. 167.
3Fang XP, Rieser MJ, Gu ZM, Zhao GX, Mclaughlin JL. Annonaceous acetogenins: An updated review. Phytochem Anal 1993;4:49-67.
4Cassady JM, Baird WM, Chang CJ. Natural products as a source of potential cancer chemotherapeutic and chemopreventive agents. J Nat Prod 1990;53:23-4.
5Sahpaz S, Gonzαlez MC, Hocquemiller R, Zafra-Polo MC, Cortes D. Annosenegalin and annogalene: Two cytotoxic mono-tetrahydrofuran acetogenins from Annona senegalensis and Annona cherimolia. Phytochemistry 1996;42:103-7.
6Sahpaz S, Laurens A, Hocquemiller R, Cave A, Cortes D. Senegalene: A novel oleifinic monotetrahydrofuranic acetogenin from seeds of Annona senegalensis. Can J Chem 1994;72:1533-6.
7You M, Wickramaratne DB, Silva GL, Chai H, Chagwedera TE, Farnsworth NR, et al. (-)-Roemerine, an aporphine alkaloid from Annona senegalensis that reverses the multidrug-resistance phenotype with cultured cells. J Nat Prod 1995;58:598-604.
8Burkill HM. The plants of west tropical Africa families. 1985;1:103-5. It is a book
9Abubakar MS, Musa AM, Ahmed A, Hussaini IM. The perception and practice of traditional medicine in the treatment of cancers and inflammations by the Hausa and Fulani tribes of Northern Nigeria. J Ethnopharmacol 2007;111:625-9.
10Suleiman MM, Dzenda T, Sani CA. Antidiarrhoeal activity of the methanol stem-bark extract of Annona senegalensis Pers. (Annonaceae). J Ethnopharmacol 2008;116:125-30.
11Adams RP. Identification of essential oils by ion trap mass spectroscopy. Academic Press, INC. Harcourt Brace Jovanovich; 1989.
12Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med 1982;45:31-4.