DETERMINATION OF BIOACTIVE PHYTOCOMPONENTS FROM HYDROETHANOLIC EXTRACT OF ANNONA SQUAMOSA (LINN.) LEAF BY GC- MS

DETERMINATION OF BIOACTIVE PHYTOCOMPONENTS FROM HYDROETHANOLIC EXTRACT OF ANNONA SQUAMOSA (LINN.) LEAF BY GC- MS

S. Hemalatha, P. Amudha, N. Pushpa Bharathi and V. Vanitha ^*

Department of Biochemistry, School of Life sciences, Vels University, Chennai, Tamilnadu, India.

ABSTRACT: Annona squamosa Linn.commonly called as custard apple, is an edible tropical fruit belongs to the family Annonaceae, is said to show varied medicinal effects, including insecticide, antiovulatory and abortifacient. Hence the present investigation was carried out to determine the phytochemical composition of A. squamosa leaf extract using Gas Chromatography–Mass Spectrometry technique, while the mass spectra of the compounds found in the extract was matched with the National Institute of Standards and Technology (NIST) library. This paper shows the isolation and structural elucidation of the several new natural bioactive compounds from the leaf of A. Squamosa linn in hydroethanolic extract. GC-MS analysis expressed thirty analytes, among that two compounds were showed high peak level such as of 2-Butanone, 3-amino-4-phenyl-(10.21%) and (-)-1,2,3,4-Tetrahydroisoquinoline, 6,7-dimethoxy-2-methyl-1-(6.01%), Hence, the present study focused the leaf extract of Annona squamosa plant possessed promising source of bioactivity as well as justifying the use of this plant to treat many ailments in folk and herbal medicines.

A. squamosa, Hydroethanolic extract, GC-MS technique, Herbal medicine

INTRODUCTION: From ancient times onwards, plants have been one of the essential sources of medicines. It has been quite interest in plant based medicines, health care products, pharmaceuticals, nutrient supplements, cosmetic agents, and so forth. As per the WHO review 80% populations living in the third world countries depend solely on conventional medication for their essential human needs ¹. Plants are rich in secondary metabolites with varied pharmacological activities.  Secondary metabolites are an important source with different structural arrangements and properties ². Distinguished examples of these compounds include flavonoids, phenols, phenolic glycosides, saponins and cyanogenic glycosides ^{3, 4}.

Natural products from microbial sources have been the primary source of antibiotics, but with the increasing recognition of medicinal plants as an alternative form of health care, the screening of medicinal plants for active compounds has become very significant since it may serve as sources of antibiotic prototypes ^{5, 6}. It has been shown that in vitro screening methods could provide the needed preliminary observations necessary to select crude plant extracts with potentially useful properties for further chemical and pharmacological investigations ⁷.

Annona squamosa Linn. (Family: Annonaceae) commonly known as custard apple, is a small, woody, semi deciduos, ever green tree is cultivated throughout India for its fruits. Different parts of Annona squamosa Linn. are used in folkloric medicine for the treatment of various disease ⁸. It is considered beneficial for cardiac disease, Antidiabetic, hyperthyroidism and Antioxidant. The root is considered as a drastic purgative ⁹. An infusion of the leaves is considered efficacious in prolapsusani of children, the crushed leaves are sniffed to overcome hysteria and fainting spells, and also used for wounds and ulcer. The leaves of the plants have been used as insecticide, anthelmintic, styptic, externally used as suppurant. Fruits of Annona squamosa helpful for antidysentric and bark is used for powerful astringent, vermifuge and antidysentric.

Annonaine, an alkaloid which is found in bark and leaves of A. squamosa ¹⁰. A paste of seed powder has been applied to the head to kill lice. It is also used for destroying worm in the wound of cattles ¹¹. Leaf extract of Annona squamosa has Hypoglycemic and Antidiabetic properties. ^{12, 13} From the bark of Annona squamosa, a bioactive acetogenin with anticancer activity have been isolated ^{14, 15}. Flavonoids from leaves, aporphine, ¹⁶ alkaloids,¹⁷  glycoside ¹⁸ and squamoline were isolated from this plant. Two acetogenins, annoreticuin and isoannoreticuin, isolated from the leaves, were found to be selectively cytotoxic to certain human tumours. Leaves and stems of A. squamosa also has dopamine, coclaurine, salsolinol and alkaloids ^19-21.

Fruit is used for making milk beverages as well as ice creams. In the ayurvedic system of medicine, herbal extracts but not purified compounds have been used from centuries, because many constituents with more than one mechanism of action are considered to be beneficial. Since there is no relevant report on the phytoconstituents of Annona squamosa hydroethanol leaf extract, it was chosen for the study.

Therefore the study was initiated to determine the compounds present in the Hydroethanol leaf extract of Annona squamosa with the help of Gas Chromatography-Mass Spectroscopy technique, which may reveal an insight in its use in folk medicine.

MATERIALS AND METHODS:

Collection of Sample: During the months from November to January, fresh leaves of Annona squamosa plant was collected.

Authentification of the Plant Material: The taxonomic identification of the plant material was authenticated by Prof. P. Jayaraman, Director, Plant Anatomy Research Centre, Chennai, India. A voucher specimen is maintained in plant anatomy research centre, Chennai (PARC/2009/456). Fresh leaves of Annona squamosa was used for phytochemical analysis.

Preparation of Leaf Extract: Air dried powder was macerated with 70:30 hydroethanol and stored for 72 hours in ice cold condition. After 72 hours the extracts were filtered through a Whattman filter paper No. 42 (125 mm) and the organic layer was allowed to evaporate. The resulted dark green extracts were concentrated using a rotary evaporator with a water bath set at 40 °C. The concentrated crude extracts were lyophilized into paste (5 and 15 g respectively) and were taken for further investigation.

Gas Chromatography- Mass Spectrum Analysis (GC-MS): GC-MS technique was used in this study to identify the phytocomponents present in the Annona Squamosa leaf extract. GC-MS technique was carried out at Sargam laboratory, Chennai, Tamil Nadu. GC-MS analysis of this extract was performed using GC SHIMADZU QP2010 system and gas chromatograph interfaced to a Mass Spectrometer (GC-MS) equipped with Elite-1 fused silica capillary column of 30 m length, 0.25mm diameter and 0.25 μm thickness and composed of 100% Dimethyl poly siloxane. For GC-MS detection, an electron ionization energy system with ionization energy of 70eV was used. Helium gas (99.999%) was used as the carrier gas at a constant flow rate of 1.51ml/min and an injection volume of 2μl was employed. Injector temperature was 200 °C and Ionsource temperature was 200 °C.

The oven temperature was programmed from 70 °C (isothermal for 2 min.), with an increase of 300 °C for 10 min. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds with scan range of 40 – 1000 m/z. Total GC running time was 35 min. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was a GC MS solution ver. 2.53.

Identification of Components: Interpretation of mass spectrum GC-MS was conducted using the database of National Institute Standard and Technique (NIST08s), WILEY8 and FAME having more than 62000 spectral patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST08s, WILEY8 and FAME library. The Name, Molecular weight, Molecular formula and Structure of the component of the test material was ascertained.

RESULTS AND DISCUSSION:

GC-MS Analysis: In the present study, thirty compounds have been identified from hydroethanol extract of the leaves of Annona squamosa by GC-MS analysis. The chromatogram obtained by hydroethanol fraction of Annona squamosa leaf extract was shown in Fig. 1. The active principle, area of the peak, Concentration (%) and Retention Time (RT) are presented in Table 1 and the Bioactivity of phytocomponents are presented in Table 2. Fig. 2 shows the structure of identified phytochemical compositions. The prevailing compounds were 2-Butanone, 3-amino-4-phenyl-(10.21%), (-)-1,2,3,4-Tetrahydroisoquinoline, 6,7-dimethoxy-2-methyl-1-(6.01%), Octadecanoic acid (2.90%), Resorcinol(2.37%), n-Hexadecanoic acid (2.62%),  Oleic acid(1.64%), Phenol(1.11%), 9, 12, 15 - Octadecatrienoic acid (Z,Z,Z)-, 2-Methoxy-4-vinylphenol(0.86%),  dl-à-Tocopherol (0.63%).

9,12,15-Octadecatrienoic acid, (Z,Z,Z) has the property of anti-inflammatory, insectifuge, hypocholesterolemic, cancer preventive, nematicide, hepatoprotective, antihistaminic, antieczemic, antiacne, 5-alpha reductase inhibitor, antiandrogenic, antiarthritic and anticoronary properties ²².  n-Hexadecanoic acid has the property of antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant activities ²³. Oleic acid are found in plants and animals are  used to produce hormone-like substances that regulate a number of  functions including blood clotting, blood pressure, blood lipid levels, immune responses and the inflammation responses to injury infection ²⁴. The study concludes that the extraction of hydroethanol produces number of active constituents responsible for many biological activities. So the compounds can be used for the development of folklore medicines and further experiments need to be undertaken to determine its bioactivity and to elute novel active compounds from Annona squamosa plants which may be created a new way to treat many incurable diseases. (Dr. Duke's Phytochemical and Ethnobotanical Database).

FIG. 1: CHROMATOGRAM OBTAINED FOR HYDROETHANOL FRACTION OF ANNONA SQUAMOSA LEAF EXTRACT

TABLE 1: THE PHYTOCHEMICAL COMPOSITION OF HYDROETHANOLIC EXTRACT OF ANNONA SQUAMOSA LEAF

TABLE 2: BIOACTIVITY OF PHYTOCOMPONENTS IDENTIFIED IN THE HYDROETHANOL EXTRACTS OF ANNONA SQUAMOSA (LINN.) LEAF BY GC‑MS

Phenol

Oxazolidine, 2,2-diethyl-3-methyl-

Ethanone, 1-(2-methyl-1-cyclopenten-1-yl)-

Resorcinal

2-Methoxy-4-vinylphenol

Phenol, 3,4-dimethoxy-

6,10-Dodecadien-1-yn-3-ol,3,7,11-trimethyl-

2-Butanone, 3-amino- 4-phenyl-

Dinocap

Oleic Acid

Acetic acid, 2-(2,2,6-trimethyl-7-

1H-1,2,3-Triazole oxa-bicyclo[4.1.0]hept-1-yl)-

9,12,15-Octadecatrienoic acid,  (Z,Z,Z)-

9-Octadecenoic acid, (2-phenyl -1,3- dioxolan-4-yl)methyl ester,

(-)-1,2,3,4-Tetrahydroiso quinoline,6,7-dimethoxy -2-methyl-1-

dl-à-Tocopherol

FIG. 2: THE STRUCTURES OF IDENTIFIED PHYTOCHEMICAL COMPOSITIONS

REFERENCES:

1. Vanitha V, Umadevi KJ, Vijayalakshmi K: Determination of Bioactive Components of Annona squamosa L Leaf by GC- MS Analysis. International Journal of Pharmaceutical Sciences and Drug Research 2011; 3(4): 309-312.
2. de-Fatima A, Modolo LV, Conegero LS, Pilli RA, Ferreira CV, Kohn LK, de-Carvalho JE: Lactones and their derivatives: biological activities, mechanisms of action and potential leads for drug design. Current Medicinal Chemistry 2006; 13: 3371-3384.
3. Shahidi F: Antioxidant factors in plant foods and selected oilseeds. BioFactors 2000; 13: 179-185.

4. Shahidi F, McDonald J, Chandrasekara A, Zhong Y: Phytochemicals of foods, beverages and fruit vinegars: chemistry and health effects. Asia Pacific Journal of Clinical Nutrition 2008; 17: 380-382.
5. Meurer-Grimes B, Mcbeth DL, Hallihan B, Delph S: Antimicrobial activity in medicinal plants of the Scrophulariaceae and Acanthaceae. International Journal of Pharmacognosy 1996; 34: 243-248.
6. Koduru S, Grierson DS, Afolayan AJ: Antimicrobial activity of Solanum aculeastrum. Journal of Pharmaceutical Biology 2006; 44: 283-286.

7. Mathekaga AD, Meyer JJM: Antibacterial activity of South African Helichrysum South African Journal of Botany 1998; 64: 293-295.
8. Suresh K, Mamoharan S, Panjamurthy K and Kavitak: Chemopreventive and antilipidperoxidative efficiency of Annona squamosa bark extract. Pakistan journal of Biological sciences 2006; 9(14): 2600- 2605.
9. Raj Sobiya. D, Vennila Jannet. J, Aiyavu. C, Panneerselvam: The heapatoprotective effect of alcoholic extract of Annona squamosa leaves on experimentally induced liver injury in swiss albino mice. International Journal of Integrative Biology 2009; 5(3): 182-186.
10. Vohar SB, Ishwar Kumar and Naquvi SAH: Phytochemical, Pharmacological, antibacterial and anti-ovulatory studies on Annona Squamosa. Planta Med. 1975; 28: 97-100.
11. Parvin Shahnaj Mst, Islam Ekramul M, Rahman Motiur Md., & Haque Ekramul Md.: Toxicological evalution of Annotemoyin-1 isolated from Annona squamosa from long evans Rat. Pakistan Journal of Biological science 2003; 6 (18): 1593-1596.
12. Kaleem M, Asif M, Ahmed QU, Bano B: Antidiabetic and antioxidant activity of Annona squamosa extract in streptozotocininduced diabetic rats. Singapore Med J. 2006; 47(8): 670-675.
13. Gupta RK, Kersari AN, Murthy PS, Chandra R, Tandon V, Watal G: Hypoglycemic and antidiabetic effect of ethanolic extract of leaves of Annona squamosa L. in experimental animals. J ethanopharmacol. 2005; 99(1): 75-51.
14. Hopp DC, Alali FQ, Gu ZM, McLaughlin JL: Mono-THF ring annonaceous acetogenins from Annona squamosa. Phytochemistry 1998; 47(5): 803-9.
15. Hopp DC, Zeng L, Gu ZM, Kozlowski JF, McLaughlin JL: Novel mono-tetrahydrofuran ring acetogenins, from the bark of Annona squamosa, showing cytotoxic selectivities for the human pancreatic carcinoma cell line, PACA-2. J Nat Prod. 1997; 60: 581-6.
16. Seetharaman TR: Flavonoids from the leaves of Annona squamosa and Polyalthia longifolia. Fitoterapia 1986; 57: 189-198.
17. Bhakuni DS, Tewari S, Dhar MM: Aporphine alkaloids of Annona squamosa. Phytochemistry 1972; 11: 1819-1822.
18. Forgacs P, Desconclois JF, Provost R, Tiberghien et Touche, A: Un Nouvel Heteroside Nitre Extrait D’ Annona squamosa. Phytochemistry 1980; 19: 1251-125.
19. P. Khare Indian medicinal plants, An Illustrated Dictionary, Springer publication, 2008.
20. Kirtikar KR and Basu BD: In: Indian Medicinal Plant, International Book Distributors, Dehradun, India, 1999.
21. Indian Materia Medica by Dr. K.M. Nadkarni, Publisher: Bombay Popular Prakashan, reprinted: 2000.
22. Sarumathy K, Vijayayakanthia T, Dhana Rajan MS: A Protective effect of Caesalpinia sappan (CS) on acetaminophen induced Nephrotoxicity and oxidative stress in male albino J. Pharmacology and Toxilcology 2011; 1(2): 11-21.
23. Praveen kumar P, Kumaravel S, Lalitha C: Screening of antioxidant activity, total phenolics and GC-MS study of Vitex negundo. J. Biochemistry Res 2010; 4 (7): 191-195.
24. Maria Jancy Rani P, Kannan PSM, Kumaravel S: GC-MS Analysis of Lantana camara Leaves. JPRD 2011; 2(11): 63- 66.
    

    ---

    How to cite this article:

    Hemalatha S, Amudha P, Bharathi NP and Vanitha V: Determination of bioactive phytocomponents from hydroethanolic extract of Annona squamosa (Linn.) Leaf by GC- MS. Int J Pharm Sci Res 2017; 8(6): 2539-44.doi: 10.13040/IJPSR.0975-8232.8(6).2539-44.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---