GC-MS ANALYSIS AND IN VITRO ANTILEISHMANIAL ACTIVITY OF ETHYLACETATE FRACTION OF ALLIUM CEPA (EAFAC)

GC-MS ANALYSIS AND IN VITRO ANTILEISHMANIAL ACTIVITY OF ETHYLACETATE FRACTION OF ALLIUM CEPA (EAFAC)

Muhammad Anwar Panezai ¹, Jahangir Khan Achakzai^*1, Abdul Manan Kakar ¹, Ajab Khan Tareen ¹, Nazima Yousaf Khan ¹, Javed Khan ², Asmatullah Kakar ³ and Samiullah ⁴

Institute of Biochemistry ¹, Department of Zoology ³, Department of Chemistry ⁴, University of Balochistan, Quetta, Pakistan.

Department of Microbiology ², Quaid-i-Azam University, Islamabad, Pakistan.

ABSTRACT: In this study, GC-MS analysis shown that ethyl acetate fraction of Allium cepa (EAFAC) (band one) consists of 55 compounds which shown antileishmanial activity. (IC₅₀ 73.1 ± 2.1 ug/ml) is the inhibitory concentration which is observed for ethyl acetate fraction of Allium cepa (EAFAC) (band one). Leishmania major is the specie used for this leishmanicidal assay. In this bioassay, Amphotericin B (IC₅₀ = 0.29 ± 0.05 ug/ml) and pentamidine (IC₅₀ = 5.09 ± 0.09 ug/ml) were used as positive control drugs to compare the parasite inhibition with that ethyl acetate fraction of Allium cepa (EAFAC) (band one). This activity was perceived under incubation period 72 hours and incubation temperature 22°C. Methanol extract of Allium cepa (MEAC), Hexane fraction of Allium cepa (HFAC), Ether fraction of Allium cepa (EFAC), and Diethyl ether fraction of Allium cepa (DEFAC) and butanol fraction of Allium cepa (BFAC) exhibited no antileishmanial activities. In this research paper we represent 25 compounds out of 55 compounds of ethyl acetate fraction of Allium cepa (EAFAC) (band one).

GC-MS, Ethyl acetate fraction of Allium cepa (EAFAC) (band one), Leishmania major, Pentamidine, Amphotericin B

INTRODUCTION: The medicinal values of natural products are attaining considerable significance with the perspective of grave problem created by side effects and drug resistance or multi-drug resistance of chemically originated drugs ¹. The family of Allium consists of more than 600 members which are present in Asia, North Africa, Northern America and Europe, nutraceutically, phytochemically and biochemically are closely similar but differing in color, form and taste. All the members of this family are used as medicine, vegetables, spices and ornamentals ^{2, 3}.

Allium cepa with its complex nutritional composition is the dietary source of flavonoids in different part of the world. This medicinal plant is rich source of biologically active ingredients such as sulphur compounds, flavonoids, quercetin, fructooligosaccharides, quercetin derivatives, and kaempferols ⁴. The phytochemicals which are present in this ethnomedicinal plant extract and fractions, have exhibited biological activities for instance antihelmintic, skin diseases, diuretic, antispasmodic, expectorant, anti-infective, carminative, antibacterial, anti-inflammatory, antifungal and antioxidant activities ^{5 - 9}.

The parasitic infection, leishmaniasis, is instigated by leishmania which is a protozoan, belong to Trypanosomatidae family. Cutaneous, diffuse cutaneous, mucosal and visceral leishmaniasis are the four clinically described forms of this vector transmitted disease ^{10, 11}.

The thirty different vectors, phlebotomine, also known as sandflies which transmit more than twenty species of leishmania from one host to another ¹². The parasite of leishmaniasis, leishmania, is intracellular hemoflagellate which infects macrophages in the skin and then transfers to visceral organs and leads to visceral leishmaniasis, also known as kala-azar. The choice of drug for this communicable disease is pentavalent antimonies for instance glucantime inspite of renal and cardiac toxic effects ^{13 - 15}.

MATERIAL AND METHODS:

Plant Material: Fresh Allium cepa were collected from local market of Quetta City, Balochistan, Pakistan.

Extraction and Fractionation of Allium cepa: This research of extraction and fractionation was completed in the Institute of Biochemistry University of Balochistan Quetta. 5 kg Allium cepa were soaked into extraction containers having methanol ¹⁶. These containers containing soaked Allium cepa set aside for the duration of six days. Throughout six days container was shaken two times in 24 hours. After six days, the solvent such as methyl alcohol containing compounds extracted from Allium cepa was filtered with the help of suction filtration. The filtered methyl alcohol containing Allium cepa extract was with the help of rotary evaporator vaporized. Semisolid crude methanolic extract was removed and was 165.42 gm ^{17, 18}.

Formation of Fractions: The main extract due to methyl alcohol has been separated for instance 2 portions. 1 portion (1 gm) has been screened for leishmanicidal effect whereas 2 portions (164.42 gm) relocated in the separatory funnel for the formation of different fractions with the help of solvents such as water, n-hexane, ether, diethyl ether, ethyl acetate and butanol.

In a separatory funnel with 164.42 gm extract two solvents such as water and n-hexane have been added. With thorough shaking two layers have been created n-hexane layer and water layer. Both layers have been alienated, though; water layer has been three times extracted with n-hexane. n-hexane was with the help of rotary evaporator vaporized. Semisolid n-hexane fraction was removed and further fractionated with ether and diethyl ether resulting in ether, diethyl ether fractions.

Fractionation of n-hexane Fraction: n-hexane has been separated for instance 2 portions. 1 portion has been screened for leishmanicidal effect whereas 2 portion relocated in the separatory funnel for the formation of different fractions with the help of solvents such as ether and diethyl ether resulting in ether and diethyl ether fractions.

Formation of Ether Fraction: In a separatory funnel with n-hexane extract two solvents such as n-hexane and ether have been added. With thorough shaking two layers have been created n-hexane layer and ether layer. Both layers have been alienated, though; n-hexane layer has been three times extracted with ether. Ether was with the help of rotary evaporator vaporized. Semisolid ether fraction was removed and screened for leishmanicidal activity.

Formation of Diethyl Ether Fraction: In a separatory funnel with n-hexane extract two solvents such as n-hexane and diethyl ether have been added. With thorough shaking two layers have been created n-hexane layer and diethyl ether layer. Both layers have been alienated, though; n-hexane layer has been three times extracted with diethyl ether. Diethyl ether was with the help of rotary evaporator vaporized. Semisolid diethyl ether fraction was removed and screened for leishmanicidal activity.

Fractionation of Aquous Fraction: Aqueous fraction is further fractionated into two fractions such as ethyl acetate and butanol fractions.

Formation of Ethylacetate Fraction: In a separatory funnel with aqueous extract two solvents such as water and ethyl acetate have been added. With thorough shaking two layers have been created, aqueous layer and ethyl acetate layer. Both layers have been alienated, though; aqueous layer has been three times extracted with ethyl acetate. Ethyl acetate was with the help of rotary evaporator vaporized. Semisolid ethyl acetate fraction was removed and screened for leishmanicidal activity.

Preparatory TLC of Ethylacetate Fraction: With the help of preparatory TLC, ethyl acetate fraction was further separated into two bands such as ethyl acetate band one and ethyl acetate band two. This preparatory TLC was carried with the help of mobile phase methanol: chloroform (5:95).

Formation of Butanol Fraction: In a separatory funnel with aqueous extract two solvents such as water and butanol have been added. With thorough shaking two layers have been created, aqueous layer and butanol layer. Both layers have been alienated, though; aqueous layer has been three times extracted with butanol. Butanol was with the help of rotary evaporator vaporized. Semisolid butanol fraction was removed and screened for leishmanicidal activity. At the end of the process, different extract / fractions i.e., methanolic crude extract, n-hexane, ether, diethyl ether, butanol, ethyl acetate band one and ethylacetate band two fractions were prepared for antileihmanial activities.

Antileishmanial Bioassay: Leishmanial promasti-gotes was aseptically sedimented down at 3000 rpm for 10 min, counted with the help of improved Neubaur chamber under the microscope and diluted with the fresh medium to a final concentration of 1 × 10⁶   parasites. In a 96 well microtiter plate, 180 ul of the parasite culture (1 × 10⁶ parasites/ml) was added in different wells in which 20 µl of the experimental compound was added in culture and serially diluted so that minimum concentration of the compound was 1 µg/ml. Negative control received medium with a parasite density 1 × 10⁶ cells/ml. The positive control contained varying concentration of standard antileishmanial compound such as Amphotericin B, Pentamidine. The plate was incubated between 21-22 °C for 72 hrs. The culture was examined microscopically on an improved Neubaur chamber and IC₅₀ values of compound possessing antileishmanial activity were counted ¹⁹.

Gas Chromatography Mass Spectrometry (GC-MS) Analysis Triple Quadrupole Acquisition Method-MS Parameters:

For Identification and Quantification of Allium cepa Compounds: 2 µl of Allium cepa extract or fraction was directly injected into the gas chromatograph mod. 6890 N Network GC System (Agilent Technologies “Palo Alto, CA”) together in the presence of  mass spectrometer mod. “5973 Network Mass Selective Detector” (Agilent Technologies “Palo Alto, CA”) and furnished in the presence of “a column HP-5MS (30 m length, 0.25 mm interior diameter, 0.25 um film width” Agilent Technologies, “Palo Alto, CA”). Helium gas was off. Injection was made into a split-splitless injector (split ratio 30:1) at 250 °C. The oven program was the following: “70 °C for 3 min then 6 °C /min to 180 for 5 min, then 6 °C/min to 280 °C for 10 min, then 8 °C /min to 290 °C for 20 min”. The MSD transfer line was set at a temperature of 250 °C; MSD temperature quadrupole was of 150 °C and ionization temperature was 230 °C, Mass spectra were seventy electrovolts and scan achievement was accomplished in the series between thirty five and 300 m/z. The identification of the components of the Allium cepa extract or fraction was assigned by matching their “mass spectra with those available in the libraries NIST 02 and WILEY” ²⁰.

RESULTS:

Antileishmanial Activity of Allium cepa Fractions: Antileishmanial activity of Allium cepa extract and fractions was performed against Leishmania major. Effective antileishmanial activity was observed for ethyl acetate fraction of Allium cepa (EAFAC) while other extract and fractions such as Methanol extract of Allium cepa (MEAC), Hexane fraction of Allium cepa (HFAC), Ether fraction of Allium cepa (EFAC), Diethyl ether fraction of Allium cepa (DEFAC) and butanol fraction of Allium cepa (BFAC) exhibited no antileishmanial activities. Inhibitory concentration for ethyl acetate fraction of Allium cepa (EAFAC) observed (IC₅₀ = 73.1 ± 2.1 µg/ml). In this bioassay, Amphotericin B (IC₅₀ = 0.29 ± 0.05 µg/ml) and pentamidine (IC₅₀ = 5.09 ± 0.09 µg/ml) were used as positive control drugs to compare the parasite inhibition with that by the Allium cepa extract and fractions. This activity was perceived under Incubation period 72 h and Incubation Temperature 22 °C.

TABLE 1: ANTILEISHMANIAL ACTIVITIES OF EXTRACTS AND FRACTIONS OF ALLIUM CEPA

Chemical Composition of Ethyl Acetate Fraction of Allium Cepa (Eafac) (Band One): The ethyl acetate fraction of Allium cepa (EAFAC) (band one) which showed antileihmanial activity has been analyzed by Gas Chromatography Mass Spectrometry and results are represented in tables. Ethyl acetate fraction of Allium cepa (EAFAC) (band one) consists of 55 compounds. 31 - 55 compounds are represented in this paper. 25 compounds were evaluated by RT matching with library and MS spectra.

TABLE 2: MOLECULAR FORMULA, MOLECULAR MASS, STRUCTURE, m/z AND RT OF 31 - 35 COMPOUNDS IN ETHYL ACETATE FRACTION OF ALLIUM CEPA (EAFAC) (BAND ONE)

Compound	Molecular Formula	Molecular Mass	Structure	m/z	RT
31	C9H10O3	166		150.9	17.78
32	C2H7BO2	74		43.1	17.98
33	C8H16N2	140		43.1	18.9
34	C10H12O3	180		180	19.34
35	C7H15NO	129		56	19.85

TABLE 3: MOLECULAR FORMULA, MOLECULAR MASS, STRUCTURE, m/z AND RT OF 36 - 40 COMPOUNDS IN ETHYL ACETATE FRACTION OF ALLIUM CEPA (EAFAC) (BAND ONE)

Compound	Molecular Formula	Molecular Mass	Structure	m/z	RT
36	C6H12O5	164		60	20.64
37	C3H5N3	83		84	20.69
38	C14H18O	202		71	20.99
39	C14H14O	198		57.1	21.07
40	C8H16O	128		57.1	21.2

TABLE 4: MOLECULAR FORMULA, MOLECULAR MASS, STRUCTURE, m/z AND RT OF 41 - 45 COMPOUNDS IN ETHYL ACETATE FRACTION OF ALLIUM CEPA (EAFAC) (BAND ONE)

Compound	Molecular Formula	Molecular Mass	Structure	m/z	RT
41	C25H22O2	354		73	21.28
42	C19H36O2	296		55	21.71
43	C6H10O5	162		57.1	22.2
44	C17H34O2	270		74	22.36
45	C8H16O2	144		60	24.02

TABLE 5: MOLECULAR FORMULA, MOLECULAR MASS, STRUCTURE, m/z AND RT OF 46-50 COMPOUNDS IN ETHYL ACETATE FRACTION OF ALLIUM CEPA (EAFAC) (BAND ONE)

Compound	Molecular Formula	Molecular Mass	Structure	m/z	RT
46	C17H34O2	270		74	27.97
47	C16H32O2	256		73	28.96
48	C11H8N2	168		168	29.67
49	C18H35NO	281		59	36.77
50	C24H38O4	390		149	39.49

TABLE 6: MOLECULAR FORMULA, MOLECULAR MASS, STRUCTURE, m/z AND RT OF 51 - 55 COMPOUNDS IN ETHYL ACETATE FRACTION OF ALLIUM CEPA (EAFAC) (BAND ONE)

Compound	Molecular Formula	Molecular Mass	Structure	m/z	RT
51	C21H28OSi	324		134.9	45.27
52	C12H15DNO2	207		207	54.51
53	C24H26OSi	358		197	56.16
54	C19H23NO	281		281	62.67
55	C16H32O2Si	284		134.9	64.24

TABLE 7: MASS SPECTRA OF 31 - 35 COMPOUNDS IN ETHYL ACETATE FRACTION OF ALLIUM CEPA (EAFAC) (BAND ONE)

TABLE 8: MASS SPECTRA OF 36 - 40 COMPOUNDS IN ETHYL ACETATE FRACTION OF ALLIUM CEPA (EAFAC) (BAND ONE)

TABLE 9: MASS SPECTRA OF 41 - 45 COMPOUNDS IN ETHYL ACETATE FRACTION OF ALLIUM CEPA (EAFAC) (BAND ONE)

TABLE 10: MASS SPECTRA OF 46 - 50 COMPOUNDS IN ETHYL ACETATE FRACTION OF ALLIUM CEPA (EAFAC) (BAND ONE)

TABLE 11: MASS SPECTRA OF 51 - 55 COMPOUNDS IN ETHYL ACETATE FRACTION OF ALLIUM CEPA (EAFAC) (BAND ONE)

CONCLUSION: Finally we can conclude that Effective antileishmanial activity was observed for ethyl acetate fraction of Allium cepa (EAFAC) while other extract and fractions such as Methanol extract of Allium cepa (MEAC), Hexane fraction of Allium cepa (HFAC), Ether fraction of Allium cepa (EFAC), Diethyl ether fraction of Allium cepa (DEFAC) and butanol fraction of Allium cepa (BFAC) exhibited no antileishmanial activities. Ethyl acetate fraction of Allium cepa (EAFAC) band one consists of 55 compounds.

In this research paper we represent 25 compounds out of 55 compounds of ethyl acetate fraction of Allium cepa EAFAC (band one). In the near future, we, the researchers in the Institute of Biochemistry, University of Balochistan, Pakistan, will separate all these 55 compounds which exhibited antileishmanial activity and will lead towards drug development against leishmaniasis with least side effects.

ACKNOWLEDGEMENT: The authors are thankful to Hussain Ebrahim Jamal (HEJ), Research Institute of Chemistry, University of Karachi, Karachi, Pakistan, for providing antileishmanial facilities and GC-MS analysis and Institute of Biochemistry, University of Balochistan, Quetta, Pakistan for providing lab facilities for extraction and fractionation.

CONFLICT OF INTEREST: Authors do not have any conflict of interest.

REFERENCES:

1. Sreekanth D, Arunasree MK, Roy KR, Chandramohan Reddy Reddy GV and Reddanna P: Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosisin human chronic myeloid leukemia Cell line-K562. Phytomedicine 2007; 14: 739-746.
2. Fenwick GR and Hanley AB: Chemical composition. In: Brewster JL, Rabinowitch HD (Eds) Onion and Allied Crops, CRC Press, Boca Raton, Florida, USA 1990; 17-31.
3. Garlic and Health Group: Special section. Medicinal and Aromatic Plant Science and Biotechnology 2007; 1: 1-36
4. Roldan E, Sanchez-Moreno C, De Ancos B and Cano MP: Characterisation of onion (Allium cepa ) by-products as food ingredients with antioxidant and antibrowning properties. Food Chem 2008; 108: 907-916.
5. Griffiths G, Trueman L, Crowther T, Thomas B and Smith B: Onions--a global benefit to health. Phytother Res 2002; 16: 603-15.
6. Momeni L and Zamanzad B: The antibacterial properties of Allium cepa (onion) and Zingiber officinale (ginger) extracts on Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans isolated from vaginal specimens. J Shahrekord Univ Med Sci 2010; 11: 81-7.
7. Leung A: Encyclopedia of common natural ingredients used in food, drugs and cosmetics. New York, NY: John Wiley 1980.
8. Vohora SB, Rizwan M and Khan JA: Medicinal uses of common Indian vegetables. Planta Med. 1973; 23(4): 381–93.
9. Elnima EI, Ahmed SA, Mekkawi AG and Mossa JS: The antimicrobial activity of garlic and onion extracts. Pharmazie 1983; 38(11): 747–8.
10. Lainson R and Shaw JJ: Epidemiologyand ecology of leishmaniasis in Latin-America. Nature 1978; 273: 595-600.
11. Marsden PD: Current concepts inparasitology: New England Journal of Medicine 1979; 300: 350-352.
12. Pearson RD and Sousa AQ: Clinical spectrum of Leishmaniasis. Infect. Dis 1996; 22: 1–13.
13. Mendonca-Filho RR, Rodrigues IA, Alviano DS, Santos AL, Soares RM, Alviano CS et al.: Leishmanicidal activity of polyphenolic-rich extract from husk fiber of Cocos nucifera (Palmae). Res Microbiol. 2004; 155(3): 136–43.
14. Desjeux P: Leishmaniasis: current situation and new perspectives. CompImmunol Microbiol Infect Dis 2004; 27: 305–18.
15. Murray HW, Berman JD, Davies CR and Saravia NG: Advances in leishmaniasis. Lancet 2005; 366: 1561–77.
16. Jehan B, Khan S and Shafi M: Antimicrobial potentials of fresh Allium cepa against gram negative bacteria and fungi. Pak. J. Bot 2013; 45: 1-6.
17. Achakzai JK and Anwar M: GC-MS Analysis and Antileishmanial activity of dichloromethane fraction of Allium cepa (DFAC) in vitro. International Journal of Pharma and Bio Sciences 2016; 7(2): 40-51.
18. Achakzai JK, Panezai MA, Kakar AM and Khan J: In vitro Leishmanicidal Effect and Gas Chromatography Mass Spectrometry analysis of Chloroform fraction of Allium cepa. European Academic Research 2016; 4(3): 2678-2691.
19. Rahman A, Chaudhary MI and Thompson W: Bioassay Techniques for Drug, Harwood Academic publishers, The Netherland 2001; 60.
20. El-Wakil EA, El-Sayed MM and Lateef EESA: GC-MS Investigation of Essential oil and antioxidant activity of Egyptian White Onion (Allium cepa). International journal of pharma sciences and research. 2015; 6: 3.
    

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    How to cite this article:

    Panezai MA, Achakzai JK, Abdul Manan Kakar AM, Tareen AK, Khan NY, Khan J, Kakar A and Samiullah: GC-MS analysis and in vitro antileishmanial activity of ethylacetate fraction of Allium cepa (EAFAC). Int J Pharm Sci Res 2018; 9(4): 1600-08.doi: 10.13040/IJPSR. 0975-8232.9(4).1600-08.

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