Morinda citrifolia L. (Noni) as a free radical scavenger and an anticancer agent

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1 Research Article Morinda citrifolia L. (Noni) as a free radical scavenger and an anticancer agent Jesse Joel T*, Jagdish Kumar Suluvoy, R. Hiroshini Raja ABSTRACT Aim: The aim of this study was to explore, extract, and validate the leaves of Morinda citrifolia L. plant for the presence of phytochemicals against KB3-1 oral human cancer cell lines. Materials and Methods: In the current investigative study, the methanol extract of leaves of M. citrifolia L. a traditional Polynesian medicinal plant (180 mg/kg bd.wt) which was taxonomically identified and authenticated was examined for its protective effect against KB3-1 oral human cancer cell lines. The antioxidant scavenging assay was done using 2,2-diphenyl-1-picrylhydrazyl, H 2, and the reference was ascorbic acid. The percentage of inhibition and regression curve for the leaf extract was plotted. Results and Discussion: The results revealed the concentration of sample required to scavenge the free radicals. The inhibitory concentration 50 value of M. citrifolia L. was obtained. The cytotoxicity studies of the leaf extract on KB3-1 oral cancer cell lines done using MTT assay reported that the extract might have potent anticancer properties. These were validated when the methanolic extract of M. citrifolia L. leaves was subjected to gas chromatography mass spectrometry, and the components with a probability of 25% and above were listed where the presence of the probable components must be the reason for the anticancer activity which gives the cytotoxic advantage to the plant in the study. Conclusion: The present exploration suggests that the methanolic extract of leaves of M. citrifolia L. has a protective effect against KB3-1 human cancer cell line and exhibits potent anticancer cytotoxicity. KEY WORDS: Anticancer, Antioxidant assay, Gas chromatography mass spectrometry, Morinda citrifolia L., Noni INTRODUCTION Morinda citrifolia L. M. citrifolia L. [Figure 1] belongs to Rubiaceae family used as an important traditional Polynesian medicinal plant and commercially known as Indian mulberry(noni), Nonu, and Cheese fruit in various cultures. [1] Its native range extends through Southeast Asia and Australia about 1300 feet above sea level. It is a small tropical evergreen shrub or tree, its trunk heights from 3 to 12 m, and distinctive grenade-like yellow fruit. The size of the fruit is about 12 cm due to coalescence of the interior ovaries of many closely packed flowers. It reaches cm in size. The fruit has a pungent odour when ripening, and is hence also known as cheese fruit. The fruit of this plant has been used as food, drink, medicine, colorful dye, and cosmetics purpose and has a high demand in medicines for different kinds of illnesses Access this article online Website: jprsolutions.info ISSN: such as diabetes, high blood pressure, AIDS, arthritis, cancer, gastric ulcer, sprains, mental depression, senility, poor digestion, atherosclerosis, and blood vessel problem due to its beneficial effects. The fruit juice is widely distributed throughout the world as nutraceutical dietary supplement. [2] Most parts of the tree have been widely used medicinally to relieve rheumatism and other pains and for their healing effects. [3] The roots, stems, bark, leaves, flowers, and fruits of the M. citrifolia L. plant are all involved in various combinations and recorded herbal remedies. Morinda is reputed to have antibacterial, antiviral, antifungal, antitumor, antitubercular effect, analgesic activity, immunological activity, and mental health and improve high frequency, antihelminthic, analgesic, hypotensive, anti-inflammatory, and immune enhancing. [2,4] Plausible Wonder Drug Cancer, a condition where cells proliferate abnormally and without control also invades adjacent cells and tissues by means of Angiogenesis and Metastasis. Studies indicate that Morinda juice may possess a preventive Department of Biotechnology, School of Agriculture and Biosciences, Karunya Institute of Technology and Sciences (Deemed to be University), Coimbatore, Tamil Nadu, India *Corresponding author: Jesse Joel T, Department of Biotechnology, School of Agriculture and Biosciences, Karunya Institute of Technology and Sciences (Deemed to be University), Coimbatore, Tamil Nadu, India. jessejoel@gmail.com Received on: ; Revised on: ; Accepted on:

2 effect of Cancer. [5] Cancer cells can also spread to other parts of the body through the blood and lymph systems. In 2012, about 165,000 children under the age of 15 have been diagnosed with cancer. The risk of cancer increases with age and more incidences occur in the developing countries. The financial costs if cancer was estimated to be at $1.16 trillion USD per year as of [6] The adverse side effects for the synthetic drugs available for cancer will affect the human and decrease the human life span. The available resources for the resource to cure the disease without any side effects are from the natural products of the plant. The novel approach is to use the natural products that reduce the side effects and toxicity. The present study or research concentrates on the leaves of the plant M. citrifolia L. and was analyzed for the free-radical activity and its anticancer properties against Human cancer cell line (KB3-1). Figure 1: A picture of Morinda citrifolia L. MATERIALS AND METHODS Plant Material and Sample Preparation The plant material was collected from the surroundings of Karunya Institute of Technology and Sciences, Coimbatore. The plant material was taxonomically identified and authenticated [Figure 2] by the Botanical Survey of India and was retained in our laboratory for future use. Preparation of Extract The plant material collected was rinsed by distilled water to remove the dust and allowed to dry under shade. After drying, it was powdered in an electric blender. Then, 180 g of the powder was suspended in 200 ml of ethanol [7] for 20 h at room temperature. The extraction process was carried out in Soxhlet apparatus. [1] The remaining solvent was evaporated by the rotary evaporator, and the extract is stored in 4 C for further use. Chemicals The chemicals and reagents were prepared freshly before the experiments conducted in laboratory grade. Phytochemical Screening The presence of phytochemicals [8] such as phenols, flavonoids, alkaloids, saponins, tannins, glycosides, and steroids was examined in the extract. The qualitative results were expressed for the presence and absence of the phytochemicals. Detection of Phenols and Tannin Few drops of 10% ferric chloride were added with 0.3 ml of extract which gives blue or green color precipitate due to the presence of phenol. [1,2] Detection of Flavonoids 0.3 ml of extract was treated with few drops of sodium hydroxide solution. Formation of intense yellow color, which becomes colorless on addition of dilute acid, indicates the presence of flavonoids. [1] Figure 2: Plant authenticated and identified as Morinda citrifolia L. (BSI/SRC/5/23/2018/Tech) Detection of Alkaloids Mayer s test Extracts were dissolved individually in dilute hydrochloric acid and filtered. Filtrates were treated with 0.3 ml of potassium mercuric iodide. Formation of a yellowcolored precipitate indicates the presence of alkaloids. [1] Detection of Saponins Froth test 0.3 ml of extract was added with 2 ml of distilled water in a test tube. The solution was vigorously shaken and observed for the stable froth persistence. [2] 3135

3 Detection of Glycosides (Cardiac) Keller Kiliani Test for Glycoside (Cardiac): 300μl of extract was added with 1 ml of acetic acid followed by the addition of 300μl of 10% ferric chloride and few drops of concentrated sulfuric acid along the sides of the test tubes; brownish ring and green-blue precipitate at the bottom of the test tube indicate the presence of cardiac glycoside. [2] Detection of Steroids 0.3 ml of extract was added with 1 ml of chloroform and few drops of concentrated sulfuric acid along the sides of the test tubes, and the reddish-brown color precipitate was observed at the bottom of the test tubes which indicates the presence of steroid. [2] ANTIOXIDANT ASSAY [9] 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay [10] The free radical scavenging activity [11] of the extract was measured using DPPH. A 0.1 mm solution of DPPH in methanol was prepared, and 1 ml of this solution was added to 3 ml of various concentrations ( mg/ml) of sample dissolved in methanol to be tested. After 30 min, absorbance was measured at 517 nm. Ascorbic acid was used as a reference material. [12] All tests were performed in triplicate. The scavenging activity was calculated as follows: DPPH radical scavenging activity (%) = ([Absorbance of control Absorbance of sample]/absorbance of control) 100 Hydrogen Peroxide Assay [12,13] A solution of hydrogen peroxide (20 mm) was prepared in phosphate buffer saline (PBS, ph 7.4). Different concentrations of the extract ( µg/ml) in ethanol (1 ml) were added to 2 ml of hydrogen peroxide solution in PBS. After 10 min, the absorbance was measured at 230 nm against a blank solution that contained hydrogen peroxide solution without the extract. The percentage of H 2 scavenging of the plant extract was calculated as follows: % scavenged (H 2 ) = ([Absorbance control Absorbance sample]/abs control) 100 MTT Assay or Cell Proliferation Assay The methanolic extract of M. citrifolia L. leaves was analyzed with the help of MTT assay for its anticancer properties by incorporating the extract into Kb3-1cell line. Here, 0.1 ml of the cells was seeded on a 96- well plate and incubated at 37 C for 24 h in the C incubator. After incubation, the cells were treated with the leaf extract M. citrifolia L. at different concentrations of 25, 50, 75, 100, 125, 150, 175, and 200 µg and again incubated for 24 h. Then, 10 µl of MTT (5 mg/ml) was added to each well, and the plate was incubated for 4 h. The reaction was arrested by the addition of 50 µl of dimethyl sulfoxide, and the readings were taken at 570 nm using an enzymelinked immunosorbent assay plate reader. A graph was plotted by taking the concentration of the test compound on X-axis against the percentage viability in the Y-axis. Cell survival (CS) expressed as percentage was calculated as Cell survival = (mean OD of treated cells)/(mean OD of control cells) *100 Gas Chromatography mass Spectrometry (GC- MS) Analysis on M. citrifolia L. Leaf Extract The methanolic extract of M. citrifolia L. was analyzed using GC-MS. The thermo GC-trace ultra VER: 5.0 (Bremen, Germany) and MS MSDSQ II electron mode with 70 ev ionization energy were used. The column temperature was set at about C at the rate of 6 C/min DB 35- MS capillary standard non-polar column. The GC injector was set at a temperature of 280 C and MS transfer at 290 C, respectively. The carrier gas used was helium gas having a flow rate of 1.0 ml/min. 1 µl of the sample was used for the analysis. The major compounds that are present in the leaf extract were analyzed by the retention time and the mass fragmentation patterns. The National Institute of Standards and Technology and Wiley 2.0 library was used for the detection of the compounds. RESULTS The aqueous, ethanol, and methanol extracts of M. citrifolia were screened for the presence of secondary metabolite using the standard procedure. Tests were carried out for steroid, glycoside, phenol, tannin, alkaloids, carbohydrate, flavonoids, saponin, proteins, and lipids and acidic components according to standard methods. [10] Preliminary phytochemical surveys and the knowledge of the chemical constituents of plants are desirable to understand herbal drugs and their preparations. Therefore, the phytochemical investigation of M. citrifolia fruit in the present study reveals the presence of various potential phytochemical constituents which may be useful for pharmaceutical industries and could be used as an effective nutraceutical [2] for human health. In this study, the purpose was to extract the phytochemicals in M. citrifolia L. leaves and fruits by GC-MS and highperformance thin-layer chromatography analysis. Phytochemical Analysis The preliminary phytochemical analysis [Table 1] was done on the extracts made with the help of the Soxhlet apparatus and solvent methanol was used. The methanolic extract was found to contain 3136

4 phenol, tannin, saponin, flavonoid, and glycoside phytochemicals. Hence, for further studies, the above-mentioned extract was preferred. The absence of phytochemicals such as alkaloid and steroid contains the activities of antimicrobial, anthelmintic, and antidiarrheal. However, the presence of phytochemicals such as phenol, tannin, saponin, flavonoid, and glycoside exhibits all the three activities [Table 2]. The study concentrates on the anticancer activity, for which the extract was further evaluated for GC-MS. Radical scavenging activity by DPPH method The M. citrifolia L. leaf extract showed an increased activity of DPPH radicals with the increase of concentration of the leaf extract. Ascorbic acid was used as the activities [Table 3]. Standard for determining the inhibitory concentration (IC50) value. The percentage of inhibition [Figure 3] and regression curve for the leaf extract was plotted. The IC50 value of M. citrifolia L. leaf extract was found to be IC50 values denote the concentration of sample required to scavenge 50% of the DPPH free radicals. The graph denotes that higher the concentration of the leaf extract, higher the inhibition rate [Table 4]. Radical Scavenging Activity by H 2 Method The M. citrifolia L. leaf extract showed an increased activity of H 2 radicals with the increase of concentration of the leaf extract. Ascorbic acid was used as the standard for determining the IC50 value. The percentage of inhibition [Figure 4] and Table 1: The phytochemicals present in each extract Phytochemicals Presence Absence Phenol + Tannin + Saponin + Flavanoid + Glycoside + Alkaloid + Steroid + Table 2: DPPH scavenging activity Concentration (µg/ml) Inhibition (± SD) DPPH: 2,2 Diphenyl 1 picrylhydrazyl Table 3: H 2 scavenging activity Concentration µg/ml Inhibition±SD SD: Standard deviation S.no Concentration (µg/ml) Inhibition (±SD) Figure 3: Percentage inhibition of Morinda citrifolia L. in 2,2-Diphenyl-1-picrylhydrazyl 3137

5 Figure 4: Radical scavenging activity Figure 5: MTT assay Table 4: Table depicting the radical scavenging activity of M. citrifolia L. methanol extract Concentration µg/ml Inhibition±SD M. citrifolia: Morinda citrifolia Table 5: Analysis on cancer cell line Concentration µg/ml Inhibition±SD regression curve for the leaf extract were plotted. The IC50 value of M. citrifolia L. leaf extract was found to be IC50 values denote the concentration of sample required to scavenge 50% of the H 2 free radicals. The graph denotes that higher the concentration of the leaf extract, higher the inhibition rate. Cytotoxicity Effect of Morinda citrifolia L. Methanol Extract on Human Cancer Cell Lines (KB3-1) Effect of M. citrifolia L. methanol extract on cancer cell line the methanolic extract of M. citrifolia L. leaves were analyzed with the help of MTT assay [Figure 5] for its anticancer properties towards the cancer cell line KB3-1 [Table 5]. With the increase 3138

6 Figure 6: Chromatogram of Morinda citrifolia L. methanolic extract Table 6: The compounds with more than 25% probability have been mentioned Compound name Probability Molecular formula Molecular weight Docosanoic acid, methyl ester (CAS) C 23 H Methyl stearate C 19 Pregnane 3,11,20,21 tetrol, cyclic 20,21 [(1,1 dimethylethyl) C 25 H 43 BO boronate], (3à,5à,11á,20S) (CAS) 2 Hexadecen 1 ol, 3,7,11,15 tetramethyl, [R [R*, R* (E)]](CAS) C 20 H ,5 Dimethyl 6 (1,5 dimethylhexyl) 15,16 epoxy 18 oxatetracyclo C 28 H [ (2,10).0 (5,9)]octdecane 13 one Lycoxanthin C 40 H 56 O Cyano 12 aza 1 (2) homodiamantane C 15 H 20 N Cyano 10 aza 2 (3) homodiamantane C 15 H 20 N Methyl Ester of Ricinoleic Acid C 19 9 Octadecenoic acid, 12 hydroxy, methyl ester, (Z) C 19 Methyl ester of ricinoleic acid C 19 9 Octadecenoic acid, 12 hydroxy, methyl ester, [R (Z)] C 19 Cholestan 3 one, cyclic 1,2 ethanediyl aetal, (5á) (CAS) C 29 H Cholestan 3 one, cyclic 1,2 ethanediyl aetal, (5á) C 29 H Hexadecanoic acid, methyl ester (CAS) C 17 H Hexadecanoic acid, methyl ester C 17 H Hexadecanoic acid, methyl ester (CAS) C 17 H Bromo[tri (dimethylphenylsilyl) methyl] cadmium dimer 77.7 C 25 H 33 BrCdSi Area in the concentration of the plant extract, there was an increase in the cell death. The IC50 value of the methanolic extract was found to be The graph denotes that higher the concentration, higher the inhibition rate. The IC50 value µg/ml was found to be moderate cytotoxic against the cell line KB3-1. GC-MS Analysis on M. citrifolia L. Leaf Extract GC-MS is an analytical technique that combines the separation properties of gas-liquid chromatography [Figure 6] and [Table 6] with the detection feature of mass spectrometry to identify different substances within a test sample. GC-MS was performed for the methanolic extract of M. citrifolia L. leaves. The chromatogram and the compounds have been mentioned below. DISCUSSION The compound can be further validated to exhibit potent cytotoxicity. The first reported Noni fruit juice contains a polysaccharide with antitumor activity that enhances the release of cytokine from thymocytes. 3139

7 [14] The findings from experimental pharmacological studies are of potential clinical relevance and need to be explored in detail before any recommendations can be made. A few in vitro and in vivo animal studies suggest a possible unidentified substance in unpasteurized noni fruit juice that may have a small degree of anticancer activity. The traditional and modern uses of noni are known to be antiulcer, [15] antidiabetic and antibacterial, [16] antifungal [17] etc., Along with these benefits, the clinical studies on Morinda citrifolia L. (Noni) helps in novel therapeutic products that lead to good health. [18] We can further validate exhibiting potent cytotoxicity. [19] This can be a major breakthrough and a probable anticancer drug for the treatment of the oral cancer in humans, and the isolation of the active component warrants further research. CONCLUSION This study illustrates that the leaves of the plant were extracted by Soxhlet using the solvent, and methanol has moderate anticancer activity against the oral cancer cell line KB3-1. The antioxidant assays DPPH and H 2 revealed the concentration of the sample required to scavenge the free radicals to 50% (IC50). The graph plotted exhibited that higher the concentration, higher the inhibition rate. The cytotoxicity resulted as higher the concentration of plant extract, higher the rate of cell death. The phytochemical screening was analyzed through the GC-MS and the biologically active compounds were identified in the methanolic leaf extract of Morinda citrifolia L. (Noni). REFERENCES 1. Kochuthressia K, Jaseentha M. Phytochemical investigation of active compounds in Morinda citrifolia leaves. Asian J Biochem Pharm Res 2015;5: Nagalingam S, Sasikumar CS, Cherian KM. Extraction and preliminary phytochemical screening of active compounds in Morinda Citrifolia fruit. Asian J Pharm Clin Res 2012;5: Shirodkar S, Hutchinson RL, Perry DL, White JL, Hem SL. Aluminum compounds used as adjuvants in vaccines. Pharm Res 1990;7: Pratima S, Shrikanth P. Organoleptic and preliminary phytochemical study of achchhuka (Morinda citrifolia). Int J Ayurvedic Herb Med 2015;5: Available from: [Last accessed on 2018 Oct 21]. 5. Wang MY, Su C. Cancer preventive effect of Morinda citrifolia (Noni). Ann N Y Acad Sci 2001;952: Stewart BW, Wild C. International Agency for Research on Cancer. World Cancer Report. World Health Organization; p Rivera A, Cedillo L, Castillo V, Sánchez A, Castañeda D. Bioactive constituents in ethanolic extract leaves and fruit juice of Morinda citrifolia. Sch Res Libr Ann Biol Res 2012;3: Available from: [Last accessed on 2018 Oct 21]. 8. Krishnaiah D, Nithyanandam R, Sarbatly R. Phytochemical constituents and activities of Morinda citrifolia L. In: Phytochemicals-a Global Perspective of Their Role in Nutrition and Health. Europe: InTech; Jayachitra A, Krithiga N. Study on antioxidant property in selected medicinal plant extracts. Int J Med Aromat Plants 2012;2: Available from: cabdirect/abstract/ [Last accessed on 2018 Oct 21]. 10. Singh DR, Singh S. Phytochemicals in plant parts of noni (Morinda citrifolia L.) with special reference to fatty acid profiles of seeds. Proc Natl Acad Sci India Sect B Biol Sci 2013;83: Srinivasahan V, Durairaj B. Antioxidant and free radical scavenging effect of Morinda Citrifolia fruit extract. Int J Pharm Pharm Sci 2014;6:55-9. Available from: innovareacademics.in/journal/ijpps/vol6issue4/6824.pdf. [Last accessed on 2018 Oct 21]. 12. Babu D, Gurumurthy P, Borra SK, Cherian KM. Antioxidant and free radical scavenging activity of triphala determined by using different in vitro models. J Med Plant Res 2013;7: Jayaprakasha GK, Selvi T, Sakariah KK. Antibacterial and antioxidant activities of grape (Vitis vinifera) seed extracts. Food Res Int 2003;36: Brown AC. Anticancer activity of Morinda citrifolia (Noni) fruit: A review. Phyther Res 2012;26: Srikanth J, Muralidharan P. Antiulcer activity of Morinda citrifolia Linn fruit extract. J Sci Res 2009;1: Sunder J, Singh DR, Jeyakumar S, Kundu A, De AK. Antibacterial activity in solvent extract of different parts of Morinda citrifolia plant. J Pharm Sci Res 2011;3: Available from: a06720eb1865b7be3839c04e761d3.pdf?_ga= [Last accessed on 2018 Oct 21]. 17. Kumar KT, Panda DS, Nanda UN, Khuntia S. Evaluation of antibacterial, antifungal and anthelmintic activity of Morinda citrifolia L. (Noni). Int J PharmTech Res 2009;2: Available from: abstract/ [Last accessed on 2018 Oct 21]. 18. Zin ZM, Abdul-Hamid A, Osman A. Antioxidative activity of extracts from mengkudu (Morinda citrifolia L.) root, fruit and leaf. Food Chem 2002;78: de Moraes GP, de Alencar MV, Islam MD, Islam T. Cytogenotoxic and oxidative status evaluation of Morinda citrifolia. Int Arch Med 2016;9:DOI: /1967. Source of support: Nil; Conflict of interest: None Declared 3140