INTRODUCTION:

Cancer (malignant neoplasm) is characterised by uncontrolled cellular growth, local tissue invasion, metastasis and can cause damage to essential organs¹. Cancer is one of the most fatal diseases in human population and one most frequent causes of death worldwide. To prevent the cancer, synthetic and natural sources are used in alone or combination. Medicinal plants have been in use from historical times and their use has been increasing day by day in the present world.

Naturally obtained compounds are considered safer and easily biodegradable than synthetic compounds and the problem of drug resistance seen in synthetic drugs can be easily reduced². Though chemotherapy is now being used as a standard method³, use of anti-cancer agents from natural products has increased. According to World health Organisation (WHO), greater than 80% of the total world’s population depends on the traditional medicines for satisfying their primary health care needs. The bioactive compounds like flavanoids, carbohydrates, sugars, alkaloids, saponins, tannins are associated with various pharmacological activities⁴.

Annona muricata L belongs to the family Annonaceae. It is a small tropical evergreen tree which grows about 4-6 m height^5,6. It has a widespread distribution and has its native to Central America, Caribbean and Africa⁶. All the parts of the plant have medicinal values. The fruits were dark green, spiny aggregated fruits made up of berries^7,8. The fruit can be round, oval or irregular heart shaped which is edible. Flesh is juicy, acidic, whitish and aromatic⁷. The researchers proved this miraculous fruit ten thousand times more potent than Adriamycin, a chemotherapeutic drug. This fruit effectively targets and kill malignant cells in 12 types of cancer including colon, breast, prostate, lung and pancreatic cancer. Intensive chemical investigations of the leaves and fruits of this species have resulted in the isolation of a great number of acetogenin. The isolates compounds possess pharmacological activities such as cytotoxicity, anti-oxidant, anti-bacterial and anti-fungal activities. Ripe fruit is used in preparing drinks in fever while unripe fruits serve as astringent and also in scorbutic (scurvy) conditions⁹. Leaves possess cytotoxic, anti- malarial, smooth muscle relaxant, anti-bacterial, insecticidal, anti-fungal, anti-parasitic, anti-amoebic¹⁰. The main aim of the study is to appraise the cytotoxic potential of Annona muricata fruits and leaves.

MATERIALS AND METHODS:

Collection and identification:

The fruits and leaves of Annona muricata were collected from the district of Kollam, Kerala and taxonomically identified by Dr T. Alexander, Research Associate (Environmental Science), Environmental Resource Research Centre, Peroorkada, Trivandrum.

Ash and Extractive values:

The extractives obtained by exhausting crude drugs are an indicative of appropriate measures of their chemical constituents. The alcohol soluble and water soluble extractive values^11,12 are performed according to the Indian Pharmacopeia. Ash value is a criterion to judge the identity or purity of drug part of Annona muricata fruits and leaves. Total ash is used to measure total amount of material remaining after ignition. Acid insoluble ash measures the amount of silica present, especially sand^12,13. Water soluble ash is a good indicator of either previous extraction of the water soluble salts in the drug.

Soxhlet extraction method:

The dried fruit and leaf materials were coarsely powdered and defatted with petroleum ether at 40⁰C and extracted using methanol as solvent at 60⁰C^14,15.

Phytochemical screening:

The methanolic extracts of Annona muricata fruits and leaves were subjected to various chemical tests for identification of phytochemical constituents (glycosides, saponins, alkaloids, carbohydrates, flavonoids, tannins)^11,16-19.

In-vitro cytotoxic study:

Hep G2 cell lines – MTT Assay:

MTT assay is a colorimetric assay for assessing cell viability after treating with Annona muricata fruits and leaves extract. MTT, a yellow tetrazole is reduced to purple formazan in living cells using NAD(P) H- dependent cellular oxido-reductase enzyme. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, dark purple formazan product^20,21,22. The cells are then solubilised with an organic solvent Dimethyl sulfoxide (Himedia) and the released, solubilised formazan product was measured at 540nm. Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells.

DLA and EAC cell lines – Trypan blue dye exclusion method:

This assay is based on membrane integrity of cells. Live cells possess well defined intact cell membrane, while dead cell lose their membrane integrity, thereby making pores which allow entry of dyes into the cells. Therefore, they get stained. These stained cells can be viewed by fluorescence. Varying concentrations of Annona muricata fruits and leaves extract were prepared (10-200µg/ml). The cancer cells also were aspirated from the peritoneal cavity of cancer bearing mice and were washed thrice with normal saline. The cell suspension (1×10⁶ DLA cells in 0.1ml) was added to tubes containing specific concentrations of test compounds and volume was made up to 1ml using phosphate buffer saline (PBS). Control tubes contained only cell suspension^23,24,25. The assay mixtures were incubated for 3hrs at 37⁰C and percentage of dead cells were determined by Trypan Blue Dye Exclusion method.

Haemolysis test:

Haemolysis is the rupturing of erythrocytes and release of their content in surrounding fluid. Release of haemoglobin into the extracellular matrix makes the solution red in appearance. Haemolytic activity is performed to determine the toxicity of the leaves and fruits extracts towards the normal erythrocytes. Human blood was collected in a heparinised vacutainer, centrifuged at 3000 rpm for 10min, discard the plasma and wash with normal saline. From this 2ml of the erythrocytes were transferred to 100ml volumetric flask and carefully diluted to the volume with phosphate buffer p^H 7.4. The extracts of different concentration were prepared with PBS and added with freshly prepared erythrocytes suspension. The tubes were then shaken for 30min and are allowed to stand for 6hrs at room temperature^12,26,27. The tubes were examined for the signs of haemolysis which is indicated by the reddish brown coloured solution without any deposit of erythrocytes. The results were compared with the standard haemolysis produced by the mercuric chloride.

RESULTS:

Extractive values:

The water soluble as well as alcohol soluble extractive values of Annona muricata fruit and leaves were reported in Table 1.

Table 1: Extractive values of AM leaf and fruit.

SAMPLES	WSEV (% w/w)	ASEV (% w/w)
Leaf	26.344	12.088
Fruit	74.4	40

Ash values:

The total ash, water soluble ash and acid insoluble ash values of Annona muricata was done and reported in Table 2.Graph is plotted with percentage w/w vs ash values in Fig 1.

Table 2: Ash values of AM leaf and fruit.

SAMPLES	TA(% w/w)	WSA (% w/w)	AIA (% w/w)
LEAF	8	5	1
FRUIT	7.15	1.25	0.25

Fig 1: Ash values of AM leaf and fruit.

Phytochemical screening

The preliminary phytochemical screenings of methanolic extracts of Annona muricata mainly revealed the presence of glycosides, alkaloids, carbohydrates, flavonoids and tannins (Table 3).

Table 3: Phytochemical screening of MEAML and MEAMF

TEST	MEAML	MEAMF
Glycosides
Keller – Killani test	+	+
Borntranger’s test	-	-
Saponins
Saponins	+	+
Alkaloids
Mayer’s test	+	+
Dragendroff’s test	+	+
Hager’s test	+	+
Wagner’s test	+	+
Carbohydrates
Molich’s test	+	+
Reducing sugar
Fehling’s test	+	+
Benedict’s test	+	+
Flavanoids
Alkaline reagent test	+	+
Tannins
Ferric chloride test	+	+
Lead acetate test	+	+

In-vitro cytotoxic study:

Hep G2 cell lines – MTT Assay:

In-vitro cytotoxic activity of Annona muricata fruits and leaves extracts were done in the Hep G2 cell lines by using MTT assay. The percentage cytotoxicity at various concentrations of MEAML and MEAMF were determined. The IC₅₀ values of MEAML and MEAMF were calculated as 82.53µg/ml and 12.48µg/ml respectively while comparing with Curcumin as standard (18.65µg/ml).

Fig 2: Cytotoxicity activity (MTT) of MEAML and MEAMF using Hep G2 cells

DLA and EAC cell lines – Trypan blue dye exclusion method:

In-vitro cytotoxicity of Annona muricata fruits and leaves extracts was done in DLA and EAC cell lines by using Trypan blue dye exclusion method. The percentage cytotoxicity of MEAML and MEAMF calculated at various concentrations for both DLA and EAC cell lines and a graph was plotted (Fig 5, 6) using Microsoft Office Excel 2010. IC₅₀values of MEAML and MEAMF for DLA cell lines was calculated as 140.33µg/ml and 179.49µg/ml respectively. EAC cell lines IC₅₀ was found to be 137.21µg/ml and 165.26µg/ml for MEAML and MEAMF respectively.

Fig 3: Cytotoxicity activity of MEAML and MEAMF using DLA cell lines.

Fig 4: Cytotoxicity activity of MEAML and MEAMF using EAC cell lines.

Haemolysis test:

The haemolytic activity was determined to confirm whether the Annona muricata leaves and fruits extracts shows any haemolytic activity towards the normal erythrocytes cells. The results confirms that Annona muricata leaves and fruits don’t show any significant toxicity towards the normal erythrocyte cells by comparing with the haemolysis produced by standard Mercuric chloride, indicated by reddish brown coloured solution(Fig 1).

Fig 4: Preliminary test for haemolytic activity for MEAML and MEAMF

Fig 5: Main test for haemolytic activity for MEAML.

DISCUSSION:

Preliminary studies on Annona muricata leaves and fruits were carried out for the standardisation of crude drug. Gravimetric analysis for the determination of ash value was carried out to judge the purity of crude drugs. The phytochemical evaluation of the Annona muricata leaves and fruits extracts revealed the presence of cardiac glycosides, alkaloids, saponins, carbohydrates, flavones and tannins in detectable amounts. The percentage cytotoxicity of methanolic extracts of fruits and leaves shows a dose dependent increase. The fruit extract showed a potent cytotoxic activity against Hep G2 cell lines. IC₅₀ values reveal that at a lower concentration of 12.4µg/ml fruit has an ability to kill 50% of cells than leaf (IC₅₀=85.5µg/ml) while compared with standard Curcumin (18.6µg/ml). The DLA and EAC cell lines shows dose dependent increase in plant extracts of Annona muricata. Annona muricata leaves and fruits possess no significant hemolysis which provides the evidence that the extracts do not possess any toxicity to normal erythrocyte cells. The Annona muricata leaves and fruits have potent cytotoxicity with no significant haemolysis. Allopathic medicines used in cancer chemo therapy possess severe cytotoxic activity towards normal cells. Annona muricata fruit and leaves have several advantages over allopathic drugs, like potent cytotoxic activity and doesn't show any significant haemolytic activity against normal cells.

CONCLUSION:

The present in-vitro study highlights the anti-cancer of Annona muricata leaves and fruits extract. Further in-vitro studies have to be performed in various cell lines. All the conducted experiments in the present study are based on crude extract and are considered to be preliminary and more sophisticated research is necessary to reach concrete conclusions about the findings of the present study.

ACKNOWLEDGMENTS:

We are expressing our heartfelt gratitude to Amala Cancer Research Centre, Thrissur and Biogenix Research Centre for Molecular Biology and Applied Sciences, Thiruvananthapuram for their support during the study.

CONFLICT OF INTEREST:

All authors have none to declare.

ABBREVIATIONS:

AM - Annona muricata, WSEV-Water soluble extractive value, ASEV- Acid soluble extractive value, MEAML- Methanolic extract of Annona muricata leaf, MEAMF- Methanolic extract of Annona muricata fruit, TA – Total ash, WSA – Water soluble ash, AIA – Acid insoluble ash

REFERENCE:

1. Chabner BA. Clinical strategies for cancer treatment: The role of drugs. In: Chabner B A, Longo D L, eds. Cancer Chemotherapy and Biotherapy: Principles and Practice, 4th ed. Philadelphia: Lippincott Williams & Wilkins. 2006;1–14.

2. Raj CN, Balasubramanian A. Pharmacognostic and antimicrobial studies of the leaves of Tabernaemontana divaricate. Pharmacologyonline. 2011; Vol 2:1171-1177.

3. Uma DP, Selvi S, Devipriya D, Murugan S, Suja S. Antitumour and antimicrobial activities and inhibition of invitro lipid peroxidation by Dendrobiumnobile. Afr J Biotech. 2009; 8 (10):2289-2293.

4. Duraipandiyan V, Ayyanar M, Ignacimuthu S. Antimicrobial activity of some ethnomedical plants used by Paliyar tribe from Tamil Nadu, India. BMC Compl Alter Medi. 2006; pp: 635.

5. Onyechi, Uchenna A, Ibeanu, Nkiruka V, Eme, Eze P, Kelechi, Madubike. Nutrient, Phytochemical Composition and Sensory Evaluation OfSoursop (Annonamuricata) Pulp and Drink in South Eastern Nigeria. Int J Bas Appl Sci. 2012; 12(6):53-57.

6. Taylor L. The Healing Power of Rainforest Herbs. New York: Square One Publishers; 2005.

7. Orwa. Annona muricata. 2009. Available from URL: http://www.worldagroforestry.org/treedb2/AFTPDFS/Annona_muricata.pdf.

8. Badrie N, Schauss AG. Soursop (Annona muricata L.): Composition, Nutritional value, Medicinal Uses and Toxicology. In: ed Watson R R, Preedy V. Bioactive food in promoting Health. Oxford: Academic press; 2009; pp:621-643.

9. Nandakarni KM. Indian MateriaMedica, Vol-1. 3rd edition. Bombay: Popular Prakashan. 1996; pp: 115.

10. Prajapati, Purohit, Sharma, Kumar.A Hand book of medicinal plants – A complete source book, Section II: Medicinal Plants A-Z. India: Agrobios.2003; 50-51.

11. IPC. Indian Pharmacopoeia, Vol 1. 5^th edition. Ghaziabad: Indian Pharmacopoeia Commission.2007; pp:198.

12. Mukherjee P K. Quality Control of Herbal Drugs: An Approach to Evaluation of Botanicals. New Delhi:Business Horizons.2002;pp: 189-192, 212-214.

13. Kokate CK, Purohit AP, Gokhale C.Pharmacognosy, Vol 1. 47th edition. Delhi: Nirali Prakashan.2013;pp: 7.4, 7.5, A.1-A.5.

14. Kannan S,Aravinth S, Jesuraj V, Kumar ESJ, SaminathanK, Suthakaran R, Kumar MR, Devi BP. Wound Healing Activity of Mimosa pudica Linn Formulation. Int J PharmaTech Res. 2009; 1(4):1554-1558.

15. Saboo SS, Thorat PK, Tapadiya GG, Khadabadi SS. Evaluation of Phytochemical and Anticancer Potential of Chloroform Extract of Trichosanthestricuspidatalour roots (Cucurbitaceae) using Invitro models. Int J Pharm Pharma Sci. 2013; 5(4):203-208.

16. Jaliwala YA, Panda PK, Patro VJ, Neha C, Kumar BN, Pandit A, Mohanty PK. Pharmacognostic and Preliminary Phytochemical screening of Ficusarnottianamiq. J Cur Pharma Res. 2011; 6 (1): 21-27.

17. Khandelwal KR. Preliminary Phytochemical screening, Practical Pharmacognosy. 6th edition. Pune: Nirali Prakashan.2006; pp: 149-539.

18. Ferguson NM. A Textbook of Pharmacognosy. 1st edition. New York: The Macmillian company.1956;pp: 189-190.

19. Madhu CD. Plant Drug Evaluation, A Laboratory guide. 1st edition. Tamil Nadu: University press.2002;pp: 88.

20. Keser S, Celik S, Turkoglu S, Yilmaz O, Turkoglu I. Hydrogen Peroxide Radical Scavenging and Total Antioxidant activity of Hawthorn. Pharm Chem J. 2012; 2(1): 9-12.

21. Tyagi SN, Rakshit, Singh A, Raghvendra, Saxena A, Patel B D. In vitro anti-oxidant activity of methanolic and aqueous extract of FlacourtiaindicaMeri. Euro J Sci Res. 2010; 5(3): 201-206.

22. Varghese E, Sathia NS, Gopal RV, Nair A, Chittethu AB, Anson TA. Anticancer activity of chloroform extract of Helicteres isora. Int J Pharm Tech.2011; 3(2): 2560 – 64.

23. Arung ET, Wicaksono BD, Handoko YA, Kusuma IW, Yulia D, Sandra F. Anti-Cancer Properties of Diethylether Extract of Wood from Sukun ( Artocarpusaltilis ) in Human Breast Cancer (T47D) Cells. Trop J Pharma Res. 2009; 8 (4): 317-324.

24. Zaidi R, Rawat P R. Evaluation of Cytotoxicity of Food in Human Hepatoma HepG2 Cells: Comet Assay Coupled to the MTT Assay. J Nutri Food Sci. 2012; 2(6):2-5.

25. Unni RT, Shah GA, Snima KS, Kamath CR, Nair SV, Lakshmanan VK. Enhanced delivery of Phyllanthus niruri nanoparticles on prostate cancer therapy. J Bionano.2014; 8(2):101-7.

26. Prasanth NV, Dilip C, SanalDev KT, Augustine L, Saraswathi R. Evaluation of In-vitro Cytotoxic and Antioxidant activities of Ipomoea batatas. Int J Pharm Pharma Sci. 2010; 2(3): 91-92.

27. Muneerudeen J, Joshi H, Swapna D, Gururaja MP, Lekshmi P, Jipnomon J, Shastry C S. Anti-Cancer potential of Bambusabambos leaf extracts. Int Res J Pharm. 2013; 4(4): pp: 205-208.

Received on 16.03.2019 Modified on 20.04.2019

Research J. Pharm. and Tech 2019; 12(8):3802-3806.

DOI: 10.5958/0974-360X.2019.00651.6