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InternationalJournal of

Noni ResearchVolume 5 Numbers 1-2 January - July 2010

International Society for Noni Science12, Rajiv Gandhi Road, Chennai - 600 096.

Phone : 044-4901 1111 Fax : 044-4901 1149E-mail : E-mail: [email protected] Visit : www.isns.in

Editor-In-Chief

Dr. Kirti Singh

Editor

Dr. P. Rethinam

Joint Editor

Dr. T. Marimuthu

CONTENTS

Activity against Herpes Simplex Virus and cytotoxicity of Noni(Morinda citrifolia L.) leaf extractsPeriyasamy Selvam, M. Julie, Breitenbach, John C. Drach

In vitro effects of Noni fruit juice on the age-related modula-tion of proliferation of lymphocytes, IFN- production andantioxidant enzyme activities in the splenic lymphocytes ofF344 ratsFariya Yasmine, Hannah Priyanka, Uday Pundir, Aparna Mohanty,RanVijay Singh, Hima, L. and Srinivasan Thyaga Rajan

Studies on cytotoxicity activity of ethanolic fraction ofMorinda citrifolia L. (Noni) fruitPeriyasamy Selvam, T. Paul Pandi, P.Vasanth Raj

Antibacterial activities and preliminary phytochemical analysisof fruit extracts of Noni (Morinda citrifolia L.)N. Manimala, S. John Britto1, L. Joelri Michael Raj, S. Prabhu

Noni as an intercrop in CoconutGeorge V. Thomas

Insect diversity on Morinda citrifolia L. in west coast ofKeralaS.P. Shanthakumar, S. Malarvannan, V.R. Prabavathy Sudha Nair

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Activity against Herpes Simplex Virus andcytotoxicity of Noni (Morinda citrifolia L.)leaf extracts

Authors’ affiliation :1. Devaki Amma MemorialCollege of Pharmacy, Chelembra,Malapuram, India2. School of Dentistry andCollege of Pharmacy,University of Michigan,Ann Arbor,, MI, United States48109E-mail:[email protected]

Abstract : Chloroform (CMC) and ethanol (ETMC) leaf extracts ofMorinda citrifolia have been evaluated for antiviral activity against HSV byplaque reduction assay. Both extracts of the leaf of Morinda citrifolia werealso investigated for cytotoxicity in KB (oral cancer) cells. Chloroform andethanolic extracts of Morinda citrifolia exhibited inhibitory activity againstHSV at the concentration of 5 and 20 µg/ml, respectively. Chloroform andethanolic extracts of Morinda citrifolia also were cytotoxic against KB cellsat the concentration of 45 and 100 µg/ml respectively.

Periyasamy Selvam1

Julie M. Breitenbach2

John C. Drach2

Correspondence to :1. Devaki Amma MemorialCollege of Pharmacy, Chelembra,Malapuram, India

Keywords : Morinda citrifolia, Noni, KB cells, HSV, HFF cells.

Introduction

The development of antiviral drugs has provided crucial new means to mitigate orrelieve the debilitating effects of many viral pathogens. A rich source for the discoveryof new HSV infection inhibitors has been and continues to be, the 'mining' of thelarge diversity of compounds already available in nature and specifically those frombotanical extracts. Morinda citrifolia L (Noni) is a versatile medicinal plant with abroad spectrum of pharmacological activities. Morinda citrifolia is reported topossess hepatoprotective (Wang et al., 2008a,b) anticancer (Akihisa et al., 2008),immunomodulatory (Palu et al., 2008), anti-inflammatory (Palu et al., 2007),wound healing (Nayak et al., 2007), antioxidant(Su et al., 2005), anti-tubercular(Saludes et al., 2002) and wide spectrum of biological activity (Pawlus and Kinghorn2007) and safe herbal drug (West et al.,2006). Recently much attention has beendevoted to searching for potential antimicrobial agents from natural products butanti-HSV activity of Morinda citrifolia is relatively less explored. The present workis to study the antiviral activity against herpes simplex virus (HSV) of chloroform andethanol extracts of the leaf powder of Morinda citrifolia and cytotoxicity were alsostudied in KB cells.

Materials and Methods

Morinda citrifolia leaf were collected from Chelembra, Malapuram District of Kerala.Mature leaves of Noni selected for the extraction and leaves were washed thoroughlywith distilled water to remove impurities. Noni leaves were well dried in sun, powdered

Intl. J. Noni Res. 2010, 5 (1-2) 1

thoroughly by grinding in a mortar with a pestle and extracted with chloroform andethanol for five days by cold maceration method. After five days the solvents werefiltered and evaporated to remove the solvent completely. Dried extracts were dissolvedin dimethylsulfoxide, diluted in cell culture media to desired concentrations thenused it antiviral and anti-cancer activity assays.

Antiviral activity

Cell culture procedures. The routine growth and passage of KB and BSC cells wereperformed in monolayer cultures using minimal essential medium (MEM) witheither Hanks salts [MEM(H)] or Earle salts [MEM(E)] supplemented with 10% calfserum or 10% fetal bovine serum (HFF cells). The sodium bicarbonate concentrationwas varied to meet the buffering capacity required. Cells were passaged at 1:2 to1:10 dilutions according to conventional procedures by using 0.05% trypsin plus0.02% EDTA in a HEPES buffered salt solution as described previously (Turk et al.,1987).

Virological procedures.

The KOS strain of herpes simplex virus type 1 (HSV-1) was used in most experimentsand was provided by Dr. Sandra K.Weller, University of Connecticut. High titer HSV-1 stocks were prepared by infecting KB cells at an m.o.i. of <0.1 as detailedpreviously (Prichard et al., 1990; Turk et al., 1987). Cell growth medium waschanged every four days until cytopathology was evident in all cells. Supernatantfluids were retained as the virus stock. Virus titers were determined in BSC-1 cellsas described by Prichard et al., 1990.

Plaque reduction assays: BSCC-1 cells in 24-well cluster dishes were infected withapproximately 100 p.f.u. of HSV-1 per cm2 cell sheet using the procedures detailedabove. Following virus adsorption, compounds dissolved in growth medium wereadded to duplicate wells in four to eight selected concentrations. After incubationat 37oC for three days, cell sheets were fixed and stained with crystal violet andmacroscopic plaques were enumerated. Drug effects were calculated as a percentageof reduction in number of plaques in the presence of each drug concentrationcompared to the number observed in the absence of drug.

Cytotoxicity assays: The effect of compounds during two population doublings ofKB cells was determined by crystal violet staining and spectrophotometric quantitationof dye eluted from stained cells as described earlier (Prichard et al., 1991). Briefly,96-well cluster dishes were planted with KB cells at 3000-5000 cells per well, in atotal volume of 200 µl per well of MEM(E) plus 10% calf serum. After overnightincubation at 37oC, extracts were added in quadruplicate at six to eight concentrations.Plates were incubated at 37oC for 48 h in a CO2 incubator, rinsed with 200 µl of

2 Intl. J. Noni Res. 2010, 5 (1-2)

Periyasamy Selvam et.al. Activity against Herpes Simplex Virus and cytotoxicity of Noni (Morinda citrifolia L.) leaf extracts

HBS, and cells fixed by adding 200 µl of 95% ethanol to all plates. 100 µl per wellof 0.1% crystal violet was added, incubated for five minutes at room temperature andrinsed four times with tap distilled water. 100 µl per well of acidified ethanol wasadded and plates read at 570 nm in a spectrophotometer designed to read 96-wellELISA assay plates.

Results

The chloroform (CMC) and ethanol (ETMC) leaf extracts of Morinda citrifolia wereevaluated for antiviral activity against HSV in plaque assays. Both extracts exhibitedinhibitory activity against HSV at approximately the same concentrations of 5 to 20µg/ml, respectively (Fig. 1). Chloroform and ethanolic extracts of Morinda citrifoliaalso exhibited cytotoxicity against of KB cells (Fig. 2) at concentration of 45 to 100µg/ml.

Fig. 1. Activity of leaf extracts of Morinda citrifolia against HSV-1

Fig. 2. Anticancer activity of leaf extracts of Morinda citrifolia


Intl. J. Noni Res. 2010, 5 (1-2) 3

Discussion

Polynesians utilized the whole Noni plant in various combinations for herbal remedies(Wang et al., 2002, McClatchey, 2002) such as arthritis, diabetes, high bloodpressure, muscle aches and pains, menstrual difficulties, headaches, heart disease,AIDS, cancers, gastric ulcers, sprains, mental depression, senility, poor digestion,atherosclerosis, blood vessel problems and drug addiction. Review of literaturefound no prior studies of HSV with Noni but did reveal that three studies had beendone for anti-HIV activity:1) A compound isolated from Noni roots (1-methoxy-2-formyl-3-hydroxyanthraquinone) suppressed the cytopathic effect of HIV in infectedMT-4 cells, without inhibiting cell growth (Umezawa et al., 1992). 2). Damnacanthal(Dam), a component of noni fruit, was identified as an inhibitor of the HIV accessoryprotein Vpr induced cell death (Masakazu et al., 2006). 3) An ethyl acetate extractof stems of Noni showed moderate activity against cell line CEM-SS with IC50>2.50×102 ?g/ml and EC50 2.36×102 ?g/mL (Bina et al., 2007). From this studywe have found that chloroform and ethanolic extracts of leaf of Morinda citrifoliaexhibitst HSV activity.

Conclusion

To our best knowledge, this is the first report on the anti-HSV-1 activities of theseIndian plants locally known as Morinda citrifolia. The isolation of active compoundspossessing anti-HSV activity from Morinda citrifolia L noni is now in progress.

References

Akihisa T, Matsumoto K, Tokuda H, Yasukawa K, Seino K.I, Nakamoto K, H. KuninagaH, Suzuki T and Kimura Y.2008. Anti-Inflammatory and Potential CancerChemopreventive Constituents of the Fruits of Morinda citrifolia. J. Nat.Prod., 71(7):1322-1325.

Bina S. Siddiqui, Fouzia A. Sattar, Sabira Begum, Tahsin Gulzar, and Fayaz Ahmad.2007 Chemical Constituents from the Stems of Morinda citrifolia L. Arch PharmRes., 30 (7): 793-798,

Masakazu Kamata, Raymond P. Wu, Dong Sung Anc, Jonathan P. Saxe, RobertDamoiseauxd, Michael E. Phelps, Jing Huang, and Irvin S.Y. Chena. 2006. Cell-basedchemical genetic screen identifies dDamnacanthal as an inhibitor of HIV-1 Vprinduced cell death. Biochem Biophys Res Commun., 348(3): 1101-1106.

McClatchey W. 2002. From Polynesian healers to health food stores: changingperspectives of Morinda citrifolia (Rubiaceae). Integr Cancer Ther., 1:110-120.


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Nayak B.S, Isitor G.N, Maxwell N.A, Bhogadi V, Ramdath D. 2007. Wound-HealingActivity of Morinda citrifolia Fruit Juice on Diabetes-Induced Rats. J.Wound Care,16(2):83-87.

Palu A K,. Kim A.H, West B.J, Deng S, Jensen J, White L. 2007. Anti-inflammatory andpotential cancer chemopreventive constituents of the fruits of Morinda citrifolia(Noni). J. Nat. Prod., 70(5): 754-760.

Palu A K, Kim A.H, West B.J, Deng S, Jensen J, White L. 2008. The effects of Morindacitrifolia L. (Noni) on the immune system, its molecular mechanisms of action. J.Ethnopharmacol., 115(3): 502-507.

Pawlus A. D, Kinghorn D. 2007. A Review of the ethnobotany, chemistry, biologicalactivity and safety of the botanical dietary supplement- Morinda citrifolia (Noni).J. Pharm. Pharmacol., 59(12): 1587-1592.

Prichard M N, Prichard L E, Baguley WA, Nassiri M R, Shipman C, Jr. 1991. Three-dimensional analysis of the synergistic cytotoxicity of ganciclovir and zidovudine.Antimicrob. Aagents Chemother., 35: 1060-1065.

Prichard M N, Turk, SR, Coleman L A, Engelhardt SL, Shipman C Jr, Drach J C.1990. A microtiter virus yield reduction assay for the evaluation of antiviral compoundsagainst human cytomegalovirus and herpes simplex virus. J. Virol. Methods, 28: 101-106.

Saludes J.P, Garson M.J, Franzblau S.G, Aguinaldo A.M. 2002. Antitubercularconstituents from the hexane fraction of Morinda citrifolia L.. (Rubiaceae), PhytotherRes., 16(7):683-688.

Su B.N, Pawlus A.D, Jung H.A, Keller W J, Mclaughlin J.L, Kinghorn A.D. 2005.Chemical constituents of the fruits of Morinda citrifolia (Noni) and their antioxidantactivity. J. Nat. Prod., 68(4): 592-598.

Turk, S.R., C. Shipman, Jr., R. Nassiri, G. Genzlinger, S.H. Krawczyk, L.B. Townsend,and J.C. Drach, 1987. Pyrrolo[2,3-d]pyrimidine nucleosides as inhibitors of humancytomegalovirus. Antimicrob. Agents Chemother., 31:544-550.

Umezawa K. 1992. Isolation of 1-methoxy-2-foremyl-3-hydroxyanthraquinone fromMorinda citrifolia and neoplasm inhibitors containing the same. Japan Kokai TokyoKoho JP 06 87, 736 (94-87, 736) Appl 1992; 92/264, 311 07.

Wang M.Y, Anderson G, Nowicki D, Jensen J. 2008a. Hepatic Protection by NoniFruit Juice against CCl4-Induced Chronic Liver Damage in Female SD Rats. PlantFoods Hum. Nutr., (3), 141-147.


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Wang M.Y, Nowicki D, Anderson G, Jensen J, West B. 2008b. Liver protective effectsof Morinda citrifolia (Noni). Plant Foods Hum. Nutr., 63(2): 59-63.

Wang M.Y, West B.J, Jensen C.J, Nowicki D, Su C, Palu A.K, Anderson G . 2002.Morinda citrifolia (Noni): a literature review and recent advances in Noni research.Acta Pharmacol Sin., 23:1127-1141.

West B.J, Jensen C.J, Westendorf J, 2006. Noni Juice is Not Hepatotoxic. World J.Gastroenterol., 12(22): 3616-3620.


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In vitro effects of Noni fruit juice on theage-related modulation of proliferation oflymphocytes, IFN- production andantioxidant enzyme activities in the spleniclymphocytes of F344 rats

Authors’ affiliation :Fariya Yasmine* HannahPriyanka* Uday PundirAparna MohantyRanVijay Singh Hima, L. andSrinivasan ThyagaRajan**Integrative Medicine Laboratory,Department of Biotechnology,School of Bioengineering,SRM University,Kattankulathur -603203,Tamil Nadu*Authors equally contributed tothis workEmail:[email protected]

Abstract : Roots, barks, and fruits of the plant Morinda citrifolia (Noni)have been used for the treatment of a number of diseases. A wide rangeof phytochemicals present in the Noni are responsible for its antibacterial,antiviral, antifungal, antitumor, antihelminthic, analgesic, hypotensive, anti-inflammatory, and immune enhancing effects. Several studies from ourlaboratories have established the relevance of communication between thenervous system, endocrine system, and immune system in maintenance ofhealth and the onset of diseases and cancer. The purpose of the study wasto determine the effects of Noni fruit juice (shelf juice, fruit juice withseeds, and fresh juice without seeds) on the proliferation of lymphocytesand antioxidant enzyme [superoxide dismutase (SOD), glutathione peroxidase(GPx), catalase, and glutathione-s-transferase (GST)] activities in thesplenocytes from young (3 mo), middle aged (8-9 mo) and old (18-22mo) F344 rats. Lymphocytes isolated from were treated with Noni juiceand Con A-induced lymphocyte proliferation was assessed. The effect ofNoni on Con A-induced production of cytokine levels (IL-2 and IFN- ) isalso being assessed. Noni Shelf juice and Noni juice with seeds augmentedantioxidant enzyme activities (significantly increased catalase, GPx, GST andSOD activities (p<0.05)), reduced lipid peroxidation (p<0.05) and increasedproliferation of lymphocytes in both the young and middle-aged rats.However, in the old animals there was no significant increase in proliferationin response to Con A stimulation. Noni shelf juice was found to augmentproliferation of the lymphocytes (p<0.05) in all the three age groups incomparison to controls. However, treatment of Con A-induced lymphocyteswith Noni juice with seeds increased lymphocyte proliferation in middleaged rats (p<0.05). In contrast, treatment with Noni juice without seeds,showed no significant difference from the control group. Taken together,Noni juice (Shelf juice and fruit juice with seeds) enhances the lymphocyticantioxidant enzyme activities in young and middle-aged rats whereas Noni

Keywords : Cytokine, superoxide dismutase, catalase, glutathione peroxidise,spleen, aging.

Fariya Yasmine*Hannah Priyanka*Uday PundirAparna MohantyRanVijay SinghHima, L. andSrinivasanThyagaRajan**

Correspondence to :Srinivasan ThyagaRajanIntegrative Medicine Laboratory,Department of Biotechnology,School of Bioengineering,SRM University,Kattankulathur -603203,Tamil Nadu

Intl. J. Noni Res. 2010, 5 (1-2) 7

Introduction

Various parts of the plant Morinda citrifolia (Noni) including the roots, barks, andfruits have been used for the treatment of a number of diseases for the past severalcenturies. The spectrum of its health benefits range from antibacterial, antiviral,antifungal, antitumor, antihelminthic, analgesic, hypotensive, anti-inflammatory, andimmune enhancing effects (Potterat et al., 2007; Wang et al., 2002). These effectsare believed to be exerted through a number of phytochemicals such asanthraquinones in roots, and iridoids, fatty acid glycosides and alcohols in fruits.Several studies from others and our laboratories have provided a functional basisfor the link between the three homeostatic systems namely, nervous system, endocrinesystem, and immune system (ThyagaRajan and Felten, 2002). A number of studieshave provided evidence for the existence of a bi-directional communication betweenthe central nervous system (CNS) and the immune system (ThyagaRajan and Felten,2002). Many of the psychosomatic diseases have autoimmune phenomena in theiretiology, a number of stressors precipitate the pathogenesis of neoplasms, andpsychosocial factors such as depression, marital separation, divorce, and bereavementare associated with immunosuppression. A number of areas in the brain, especiallyhypothalamus, respond to immune molecules through alterations in the metabolismof neurochemicals. However, the interaction and cross-regulation between thesethree systems are poorly understood. Although the beneficial effects of Noni havebeen reported through few studies (Palu et al., 2008), the mechanism(s) of action(s)from the perspective of the neuroendocrine-immune network have not beeninvestigated. Therefore, the present study was conducted to assess the effects ofMorinda citrifolia (Noni) on the age-related modulation of antioxidant status andproliferation of lymphocytes isolated from the spleen of young (3 mo), middle aged(8-9 mo) and old (18-20 mo) male F344 rats.


Animals

Young (3 mo), middle aged (8-9 mo) and old (18-20 mo) F344 rats were obtainedfrom the National Institute of Nutrition at Hyderabad and for the study. The animalswere acclimatised to the animal house at SRM University for a period of one week.They were sacrificed by decapitation at 08:00 hrs and the spleens were dissectedinto falcon tubes containing sterile ice cold HBSS containing sodium bicarbonateand Hepes.

Fariya Yasmine et.al. In vitro effects of Noni fruit juice on the age-related modulation of proliferation oflymphocytes, IFN production, and antioxidant enzyme activities in the splenic lymphocytes of F344 rats

juice without seeds did not exert any significant effects. These propertiesof Noni fruit juice may be responsible for its immune-modulating properties.(Funded by WNRF, Chennai (Grant no.: FFEP-CRP-08-002)

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Isolation of Lymphocytes

Splenocytes were isolated by first dissociating the spleen tissues using a stomacherlab blender. The cell suspensions were then passed through a fine nylon mesh toremove large aggregates and washed once in HBSS. Erythrocytes were removed bylayering the cell suspension on Histopaque and centrifuging for 30 min at 2500 rpm.Cells were removed from the interface between HBSS and Histopaque and washedthrice with HBSS. After the final wash, the cells were resuspended in RPMI 1640medium supplemented with 5% fetal bovine serum, 2mM L-glutamine, 1mM sodiumpyruvate, 0.01mM nonessential amino acids, 5x105 M 2-mercaptoethanol, 100U/mlpenicillin streptomycin solution, 24mM sodium bicarbonate and 10mM Hepes bufferfor in vitro culture at 370C in a humidified with 5% CO2.

Morinda citrifolia

M. citrifolia (Noni) fruits were obtained fresh from the National Research Centrefor Noni, Salavakkam, Tamil Nadu. The fruits were stored intact for up to three daysfor use during the study. The fresh fruits were ground with and without seeds usinga blender and the pulp obtained were serially diluted in substituted RPMI mediumfrom 10% to 0.01% and a dose response curve was generated. In addition, differentdilutions of the shelf juice provided by WNRF ranging from 10% to 0.01% in RPMImedium was also used. The RPMI medium had 5% fetal bovine serum, 2mML?glutamine, 1mM sodium pyruvate, 0.01mM nonessential amino acids, 5x105 M 2-mercaptoethanol, 100U/ml penicillin streptomycin solution, 24mM sodiumbicarbonate and 10mM Hepes buffer.

Lymphocyte proliferation assay

The proliferation of lymphocytes treated with 0.1% Noni Shelf Juice, Noni juice withseeds and Noni juice without seeds was assessed using the MTT assay in thepresence and absence of different doses of Con A ranging from 0.5 µg to 5µg/mlby maintaining appropriate controls. The concentration of Noni was determined onthe basis of preliminary studies conducted in the laboratory that enabled to establishthe dose response curve for the different juices. A 96 well plate containing 100 µlsamples having 90% confluence was treated with MTT reagent, incubated for 3 hrsand read at 620 nm after completely solubilising it in isopropanol containing 37%HCl. Triplicate wells were used for each experimental condition. Plated cells wereincubated in the presence of 5% CO2 at 37oC for 72 hrs. Replacement of mediumcontaining Noni fruit dilutions was performed every day.

Cytokines

Cells were plated in a 24 well plate and incubated with 1.25 µg/ml Con A for 24hrs in the presence of 5% CO2 at 37°C and IFN- was measured using ELISA kit(R&D Biosciences).

Intl. J. Noni Res. 2010, 5 (1-2) 9


Antioxidant enzyme Assays

The activity of antioxidant enzymes including catalase, glutathione peroxidase,superoxide dismutase and thiobarbiturate assay for extent of lipid peroxidation weredone on the basis of established protocols as shown below.

a) Estimation of catalase : The total catalase activity was measured. To 0.1 ml of thesample, add 0.9ml of phosphate buffer, shake well. From this solution, 0.5 ml wastaken, added 1 ml of hydrogen peroxide solution and the optical density was readat 240 nm at 30-second intervals for 1 minute in a spectrophotometer. The totalcatalase activity was expressed in units/ min/ mg of protein.

b) Estimation of Lipid peroxidation : The extent of lipid peroxidation was measuredin terms of rate of formation of adducts with thiobarbituric acid according to themethod of Yagi (1976). To 0.1 ml of the sample, added 0.2 ml of ice-cold 10%trichloroacetic acid, incubated for 15 min and centrifuged at 2200g for 15 min. Thesupernatant (0.2 ml) is then added to equal volume of 0.67% TBA and incubatedin a boiling water bath for 10 min. After this the samples are cooled and read at530 nm in a spectrophotometer. Standard Curve is plotted using MDA dilutions. Theresults are expressed in terms of MDA equivalents/min/mg protein.

c) Estimation of superoxide dismutase activity : The superoxide dismutase activitywas measured using the method of Saggu et. al., 1989. To 5 mg of tissue, added0.125 ml ethanol, 0.75 ml chloroform, cooled in ice, centrifuged and the supernatantobtained was used for the following assay. To 0.1 ml of the sample extract, added0.9 ml of carbonate buffer, 0.5 ml of distilled water, 0.5 ml of ethylene diamine tetraacetic acid, 0.5 ml of epinephrine and shaken well. The optical density was readimmediately at 480 nm in a spectrophotometer. The total superoxide dismutaseactivity was expressed in units/ min/ mg of protein. The total superoxide dismutaseactivity was expressed in units/ min/ mg of protein.

d) Estimation of glutathione peroxidase activity : To three sets of tubes containing0.1 ml of the sample, added 0.4 ml of phosphate buffer, 0.1 ml of sodium azidesolution, 0.2 ml of reduced glutathione, 0.1 ml of hydrogen peroxide, 1.0 ml ofdistilled water, shaken well and incubated for 0', 1½' and 3'; 10% trichloroaceticacid was then added, shaken well to stop the reaction and centrifuged. To 1.0 mlof the supernatant, added 4.0 ml of phosphate solution and 0.5 ml of Di-thio-nitro-benzene. The optical density was read immediately at 412 nm using aspectrophotometer. The total glutathione activity was expressed in units/min/ mg ofprotein.

Statistical Analysis

Data was analysed using ANOVA by SPSS software package. Parameters that attainedsignificance with ANOVA (P<0.05) were further analyzed by post-hoc tests.

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Fig. 1 : Effect of age on Con A-inducedproliferation of lymphocytes in male F344 rats

Fig. 2 : Effect of Noni fruit juice without seedson proliferation of lymphocytes in male F344

rats

Fig. 3 : Effect of Noni fruit juice with seeds onproliferation of lymphocytes in male F344 rats

Fig. 4 : Effect of Noni Shelf fruit juice onproliferation of lymphocytes in male F344 rats

Results

Effect of Noni juice on age-related alterations in Con A-induced proliferation oflymphocytes from F344 rats

In young and middle-aged animals, there was a dose-dependent increase inproliferation of lymphocytes isolated from spleen (Fig. 1). In both the young andthe middle aged animals, there was an increase in proliferation with 0.5 µg/ml ofCon A (p<0.05), 1.25 µg/ml of Con A (p<0.05) and 5 µg/ml of Con A (p<0.05).However, there was no increase in the proliferation of lymphocytes isolated from thespleens of old animals

Coincubation of lymphocytes with Noni juice with seeds and without seeds did notproliferation of lymphocytes in all the three age groups (Figs. 2 and 3). Ontreatment with Noni shelf juice, there was an increase in proliferation of thelymphocytes from the spleens of young and middle aged rats (p<0.05) while therewas no such pattern in the old rats (Fig. 4).

Effect of Noni juice on Con A-induced cytokine production

In young rats (Fig. 5), there was an increase in IFN- secretion upon stimulationwith Con A (p<0.05). Treatment with Noni juice with seeds, Noni seedless juice andNoni shelf juice were also accompanied by increases in IFN- production (p<0.05).In middle-aged rats, there was a significant increase in IFN- production in responseto Con-A stimulation among untreated controls and Noni shelf juice-treated

Intl. J. Noni Res. 2010, 5 (1-2) 11


Fig. 7 : Effect of Noni fruit juice on IFN-production by lymphocytes in old male F344

rats

Fig. 8 : Effect of Noni fruit juice with seeds oncatalase activity in lymphocytes in male F344

rats

Fig. 5 : Effect of Noni fruit juice on IFN-production by lymphocytes in young male

F344 rats

Fig. 6 : Effect of Noni fruit juice on IFN-production by lymphocytes in middle-aged

male F344 rats

lymphocytes (Fig. 6). However there was no significant effect of Con A on lymphocytestreated with Noni juice with seeds and Noni seedless juice. Treatment with all thethree Noni juices significantly increased IFN- production even in the absence ofcon A stimulation (p<0.05; Fig. 7). Among the old rats, con A treatment showedno significant increase in IFN- production compared to age matched controls buttreatment with Noni juices significantly elevated IFN- production (p<0.05).

Effect of Noni juice on age-related alterations in the antioxidant enzyme activities oflymphocytes from F344 rats:

Catalase: The activity of catalase was found to decline with age (p<0.05). However,treatment with 1% to 10-3% of the Noni fruit juice with seeds resulted in an increasein the catalase activity of the lymphocytes isolated from all three age groups (p<0.05;Fig. 8). The treatment of lymphocytes with 10-1 to 10-4% Noni seedless juiceshowed a significant increase in catalase activities in the case of middle-aged rats(p<0.05; Fig. 9). However, in the case of young and old rats, there was no significantdifference in the catalase activity. The treatment of lymphocytes with Noni shelf juiceshowed no significant change in the catalase activities of all the three age groups(Fig. 10).

12 Intl. J. Noni Res. 2010, 5 (1-2)


Fig. 9 : Effect of Noni fruit juice without seedson catalase activity in lymphocytes in male

F344 rats

Fig. 10 : Effect of Noni Shelf juice on catalaseactivity in lymphocytes in male F344 rats

Fig. 11 : Effect of Noni fruit juice with seedson SOD activity in lymphocytes in male F344

rats

Fig. 12 : Effect of Noni Shelf juice on SODactivity in lymphocytes in male F344 rats

Superoxide Dismutase

The activity of superoxide dismutase (SOD) was found to decline with age (p<0.05;Fig. 11). However, treatment with 1% to 10-3% of the Noni fruit juice with seedsresulted in an increase in the SOD activity of the lymphocytes isolated from all threeage groups (p<0.05; Fig. 12). The Shelf juice was also found to increase SODactivities in both middle-aged and old rats at a dilution range of 10-1 to 10-4%(p<0.05; Fig. 13). However, treatment with the seedless extract and shelf juice didnot affect the SOD activity of Young rats significantly. Similar to the activity of the shelfjuice, the seedless juice was found to increase the SOD activity in the middle agedand old rats significantly (p<0.05).

Lipid Peroxidation

With age there is a significant increase in the extent of lipid peroxidation in thelymphocytes (p<0.05). Treatment with Noni juice with seeds significantly reducesthe extent of lipid peroxidation in the middle aged and old rats compared to agematched controls (p<0.05; Fig.14). Treatment with Noni shelf juice showed asignificant decrease in the thiobarbiturate reactive substances in all the three agegroups (p<0.05; Fig. 15). On the other hand the seedless juice showed nosignificant effect on lipid peroxidation in the young and the middle aged rats.However, in the old rats, treatment with Noni seedless juice significantly increasedlipid peroxidation (p<0.05; Fig. 16).

Intl. J. Noni Res. 2010, 5 (1-2) 13


Fig. 13 : Effect of Noni fruit juice withoutseeds on SOD activity in lymphocytes in male

F344 rats

Fig. 14 : Effect of Noni fruit juice with seedson lipid peroxidation in lymphocytes in male

F344 rats

Fig. 15 : Effect of Noni Shelf juice on lipidperoxidation in lymphocytes in male F344 rats

Fig. 16 : Effect of Noni fruit juice withoutseeds on lipid peroxidation in lymphocytes in

male F344 rats

Fig. 19 : Effect of Noni fruit juice with seedson glutathione peroxidase in lymphocytes in

male F344 rats

Fig. 18 : Effect of Noni fruit juice withoutseeds on glutathione peroxidase in lympho-

cytes in male F344 rats

Glutathione Peroxidase

The glutathione peroxidase activity was found to increase significantly with age.Treatment of lymphocytes with Noni juice with seeds (Fig. 17) and Noni seedlessjuice (Fig. 18)was found to increase the GPx activity in all age groups (p<0.06).However, treatment with Noni shelf juice significantly reduces the GPx activity in allthe three age groups. (p<0.05; Fig. 19).

14 Intl. J. Noni Res. 2010, 5 (1-2)

Fig. 19 : Effect of Noni Shelf juice onglutathione peroxidase in lymphocytes in male

F344 rats


Discussion

The results from the present study demonstrate that Noni fruit juices (shelf juice andNoni with seeds) enhanced lymphocyte proliferation in the presence and absenceof Concanavalin A stimulation in young and middle aged rats. In contrast, there wasno such beneficial effect observed in the case of old rats. Treatment with Noni juicewithout seeds did not produce the enhancing effect observed with the shelf juice andNoni with seeds.

There was a concurrent increase in the IFN- production on treatment with Noniseedless, with seeds and shelf juices in young and middle-aged rats. Further, IFN-? production was also increased in the old rats after treatment with Noni juices whencompared to age-matched controls.

Unpublished data from our laboratory has shown that Noni juices increase theproliferation of lymphocytes in the case of young Wistar rats. The findings of thepresent study are also concurrent with these findings and other studies that haveshown the immune-enhancing effects of the Noni fruit extracts (Palu et al., 2008).Since plant extracts are known to have lectins that can stimulate proliferation in thelymphocytes, the increase in proliferation observed with the Noni extract could alsobe due to a lectin in the juice. The individual component (lectin and similarphytochemicals) that is responsible for the proliferative action must be ascertainedand its selective activation of immune cells must be investigated.

Treatment with Noni juice with and without seeds showed significant increase in thecatalase activities when compared to age-matched controls in middle aged and oldrats. The activity of superoxide dismutase was also found to increase on treatmentwith Noni fruit juice with seeds and Noni shelf juice in middle aged and old rats whileNoni seedless juice did not have any effect. Noni treatment (shelf juice and Noniwith seeds) significantly reduced the extent of Thiobarbiturate-reactive substancesin all the three age-groups when compared to age-matched controls suggesting thatthe phytochemicals in Noni fruit juices have the capability to suppress lipidperoxidation. However, the seedless extract did not show similar activity. Treatment

Intl. J. Noni Res. 2010, 5 (1-2) 15


with Noni juices significantly reduced the activity of glutathione peroxidase in all thethree age groups probably because of the reduction in free radicals due to thescavenging effect of Noni.

Plant extracts are known to be rich in flavonoids and other free-radical scavengersthat contribute to their antioxidant properties (Potterat et al, 2007). Thus Noni fruitjuices may contribute to enhancing the immune function by increasing the antioxidantenzyme activities, reducing the age-dependent increase in free-radicals and throughpossible lectins that may also contribute to the same effect. Further there is a strongrole played by the TH1 cytokines as shown by increases in IFN- in contributing tothis effect. It is important to examine the extent of influence exerted by Noniphytochemicals in modulating the bidirectional communication between theneuroendocrine system and immune system to alter the aging process (ThyagaRajanand Felten, 2002).

References

Palu, A.K, Kim, A.H,, West, B.J., Deng, S., Jensen, J., White, L. 2008. The effects ofMorinda citrifolia L. (Noni) on the immune system: its molecular mechanisms ofaction. J. Ethnopharmacol., 115(3):502-506.

Potterat, O. and Hamburger, M. 2007. Morinda citrifolia (Noni) fruit--phytochemistry,pharmacology, safety. Planta Med., 73(3):191-199.

Saggu, H., Cooksey, J., Dexter, D., Wells, F.R., Lees, A., Jenner, P., Marsden, C.D. 1989.A selective increase in particulate superoxide dismutase activity in parkinsoniansubstantia nigra. J Neurochem., 53(3):692-697.

Thyaga Rajan, S. and Felten, D.L. 2002. Modulation of neuroendocrine-immunesignaling by L-deprenyl and L-desmethyldeprenyl in aging and mammary cancer.Mech Ageing Dev,. 123(8):1065-1079.

Wang, M.Y., West, B.J., Jensen, C.J., Nowicki, D., Su, C., Palu, A.K., Anderson, G. 2002.Morinda citrifolia (Noni): a literature review and recent advances in Noni research.Acta Pharmacol Sin,. 23(12):1127-1141.

Yagi, K. 1976. A simple fluorometric assay for lipoperoxide in blood plasma.Biochem. Res,. 15:212-216.

16 Intl. J. Noni Res. 2010, 5 (1-2)

Studies on cytotoxicity activity of ethanolicfraction of Morinda citrifolia L. (Noni) fruit

Authors’ affiliation :Periyasamy Selvam1*T. Paul Pandi1P.Vasanth Raj21. Devaki Amma Memorial Collegeof Pharmacy, ChelembraMalapuram 673 634, Kerala, India2. Manipal College of PharmaceuticalSciences, Manipal University,Manipal 576 104, Karnataka, IndiaEmail:[email protected]

Abstract : Morinda citrifolia L (Noni) plant has been enriched withflavanoids, anthroquinone, steroids and glycosides. The present work is tostudy the cytotoxic effect of ethanolic extract and isolated fraction ofMorinda citrifolia L. fruits (MCF-ET, MCF-C-10) on HepG2 (Human LiverCancer) cell culture respectively. Noni fruits were collected from Noniresearch cum demonstration centre, Vadakkancherry, shade dried andextracted with ethanol. The active fraction of the ethanolic extract wassubjected to silica gel - column chromatography to afford purified compoundand it was identified as anthroquinone glycoside. In this study it wasattempted to evaluate the ethanolic extract and anthroquinone fraction, itsin-vitro cytotoxicity activity against HepG2 (human liver cancer) cells usingmethods like MTT and SRB assay. The ethanolic extract and isolatedfraction of Morinda citrifolia L (MCF-ET, MCF-C-10) showed very potentcytotoxicity against HepG2 cells with CTC50 (cytotoxicity 50%) values of220.42 µg/ml and 213.17 µg/ml respectively. The MCF-ET and MCF-C-10showed potent toxicity against human cancer cells from liver origin .Hence this extract merits further investigation to screen its anti canceractivity using in-vivo models.

Keywords : Morinda citrifolia, HepG2 cells, MTT, SRB.

Periyasamy Selvam1*

T. Paul Pandi1

P.Vasanth Raj2

Correspondence to :Periyasamy SelvamDevaki Amma Memorial Collegeof Pharmacy, Malapuram 676364,Kerala, India.

Introduction

The different parts of the Morinda citrifolia L (Noni) plant like fruit, root, leaf andbark have been used as hepatoprotective (Wang et al., 2008a, b), anticancer (Akihisaet al., 2008 and Philip et al., 1990), immunomodulatory (Palu et al., 2008), anti-inflammatory (Palu et al., 2007), wound healing (Nayak et al., 2007), antioxidant (Suet al., 2005), anti-tubercular (Saludes et al., 2002), wide spectrum of biological activities(Pawlus et al., 2007) and anti-HIV (Umezawa et al., 1992, Masakazu et al., 2006 andBina et al., 2007). Recently much attention been devoted for searching potential safeherbal medicines from natural products for the treatment of various diseases andMorinda citrifolia used for the treatment of a variety of diseases and safe herbal drug(West et al., 2006). Different extracts of Morinda citrifolia leaf exhibited maximumprotection against replication of HIV-1 in MT-4 cells (Selvam et al., 2008). The presentwork is to study the cytotoxicity activity of ethanolic extract and isolated fraction prepared

Intl. J. Noni Res. 2010, 5 (1-2) 17

from Morinda citrifolia fruit against HepG2 (Human liver cancer) cells by MTT andSRB assay methods.


Extraction and Isolation

Morinda citrifolia fruits were collected from Noni Research cum demonstrationcentre, Vadakkancherry, Thirussur District, Kerala, India, fresh fruits were cut intosmall pieces, shade dried at room temperature and then powdered. Dried fruitpowder (400gm) was subjected to hot continuous extraction in Soxhlet extractorwith ethanol for 72 hours. After extraction the solvent was distilled out and syrupyextract was concentrate under the vaccuo and kept in a desicator. Active constituentswere separated from dried ethanolic extract (MCF-ET) by using columnchromatography a solvent system of CHCl3-MeOH (95:5, 90:10 and 85:15) andincrease the polarity by gradient elution. All fractions were monitored by TLC (ThinLayer Chromatography) and spots are visualized by iodine chamber. CompoundMCF-C-10 was identified as anthroquinone glycoside by Borntrager's test. Ethanolicextract (MCF-ET) and isolated fraction (MCF-C-10) was studied on cytotoxicityagainst HepG2 (Human liver cancer) cells by using MTT and SRB assay methods.

Preparation of suspensions

Ethanolic extract (MCF-ET) and isolated fraction (MCF-C-10) of Noni fruits (Morindacitrifolia L) dissolved in DMSO and the volume was made up to 10ml with DMEM/MEM to obtain a stock solution of 1mg/ml concentration and stored at -20°C priorto use. Further dilutions were made to obtain different concentrations ranging from1000-62.5 µg/ml with respective media and used for in vitro investigations.

Cell lines and growth media

HepG2 (Human liver cancer) cells were cultured in MEM (Minimum EssentialMedium) and DMEM (Dulbecco's Modified Eagles Medium) respectively. The mediumalso contains 10% fetal calf serum, penicillin (100 IU) and streptomycin (100 µg).

In vitro cytotoxicity screening

The ability of the cells to survive a toxic insult is the basis of most cytotoxicity assays.The monolayer cell culture was trypsinized and the cell count was adjusted to1.0x105 cells/ml using medium containing 10% new born calf serum. To each wellof the 96 well microtiter plate, 0.1ml of the diluted cell suspension (approximately10,000 cells) was added. After 24 hours partial monolayer was formed, the supernatantliquid was flicked off washed the monolayer once and 100 l of different drugconcentrations were added to the cells in microtiter plates. The plates were thenincubated at 37oC for 3 days in 5% CO2 atmosphere, and microscopic examination

Periyasamy Selvam et. al. Studies on cytotoxicity activity of ethanolic fraction of Morinda citrifolia L. (Noni) fruit

18 Intl. J. Noni Res. 2010, 5 (1-2)

was carried out and observations recorded every 24 hours. After 72 hours, the drugsolutions in the wells were discarded and MTT and SRB assays performed.

Results

Ethanolic extract (MCF-ET) and isolated fraction (MCF-C-10) of Noni fruits (Morindacitrifolia L) showed very potent cytotoxicity against HepG2 cells with CTC50(cytotoxicity 50%) values of 200µg/ml, 220µg/ml and 246µg/ml 213µg/ml respectively(Table 1). Methods like MTT and SRB assay followed. MCF-ET and MCF-C-10showed potent toxicity against human cancer cells from liver origin respectively. Inthe present study ethanolic extract and isolated compound exhibited same potencyof anticancer activity. This in vitro study has proved the selective toxicity Morindacitrifolia against cancer cells.

Table 1. Determination of CTC50 by using MTT and SRB assay in HepG2(Human liver cancer) cell cultures

Extract Method CTC50

* value (in µg/ml)

MCF-ET MTT 200.42 ± 4.31

MCF-ET SRB 220.2 ± 11.93

MCF-C10 MTT 246 ± 5.88

MCF-C10 SRB 213 ± 7.74

* Cytotoxic 50% concentration

Discussion

The Polynesians utilized the whole Noni plant (Morinda citrifolia) in variouscombinations for herbal remedies and reported to possess wide spectrum biologicalactivities such as arthritis, diabetes, high blood pressure, muscle aches and pains,menstrual difficulties, headaches, heart disease, AIDS, cancers, gastric ulcers, sprains,mental depression, senility, poor digestion, atherosclerosis, blood vessel problemsand drug addiction (Wang et al., 2002 and McClatchey et al., 2002). The differentextracts of stem and fruits of Morinda citrifolia L were screened for their anti-HIVactivity and cytotoxicity, some of this extracts exhibited anti-HIV activity against HIV-1 and marked cytotoxicity in MT-4 cells (Selvam et al., 2007 and 2009). Reviewof the literature revealed that anticancer activity of Morinda citrifolia (Noni) is dueto the anthroquinone and flavanoids (Philip et al., 1990 and Palu et al., 2008). Inthe present study ethanolic extract and isolated fraction of anthroquinone compoundexhibited same potency of anticancer activity. Ethanolic extract and isolated fraction


Intl. J. Noni Res. 2010, 5 (1-2) 19

of Noni fruit showed potent toxicity against HepG2 (human cancer cells) from liverorigin . This extract merits further investigation to screen its anti-cancer activity usingin vivo models and further studies regarding isolation of active constituents fromethanolic extract are under way.

References

Akihisa, T., Matsumoto, K., Tokuda, H., Yasukawa, K., Seino, K.I., Nakamoto, K. H.Kuninaga, H., Suzuki, T. and Kimura, Y. 2008. Anti-inflammatory and potentialcancer chemopreventive constituents of the fruits of Morinda citrifolia. Journal ofNatural Product, 71:1322-1325.

Bina, S. Siddiqui., Fouzia, A. Sattar, Sabira Begum, Tahsin Gulzar, and Fayaz Ahmad.2007. Chemical constituents from the stems of Morinda citrifolia Linn. AchievePharmaceutical Research, 30 (7): 793-798.

Francis, D and Rita L. 1986. Rapid colorimetric assay for cell growth and survivalmodifications to the tetrazolium dye procedure giving improved sensitivity andreliability. Journal of Immunological Methods, 89: 271-277.

Masakazu Kamata, Raymond, P Wu, Dong Sung Anc, Jonathan, P Saxe, RobertDamoiseauxd, Michael E Phelps, Jing Huang and Irvin S.Y. Chena. 2006. Cell-basedchemical genetic screen identifies damnacanthal as an inhibitor of HIV-1 Vpr inducedcell death. Biochemical Biophysics Research Communication, 348 (3): 1101-1106.

McClatchey, W. 2002. From Polynesian healers to health food stores changingperspectives of Morinda citrifolia (Rubiaceae). Integrated Cancer Therapy, 1:110-120.

Nayak BS, Isitor GN, Maxwell NA, Bhogadi V and Ramdath D. 2007. Wound-HealingActivity of Morinda citrifolia Fruit Juice on Diabetes-Induced Rats. Journal WoundCare, 16 (2) :83-87.

Palu, A.K/, Kim, A.H., West BJ, Deng S, Jensen J and White L.2007. Anti-Inflammatoryand Potential Cancer Chemopreventive Constituents of the Fruits of Morinda citrifolia(Noni). Journal of Natural Product, 70 (5): 754-760.

Palum, A.K., Kim, A.H., West, B.J., Deng, S., Jensen, J. and White, L. 2008. The Effectsof Morinda citrifolia L. (Noni) on the Immune System, Its Molecular Mechanismsof Action. Journal of Ethnopharmacology, 115 (3): 502-507.

Pawlus, A.D and Kinghorn, D. 2007. A Review of the ethnobotany, chemistry, biologicalactivity and safety of the botanical dietary supplement - Morinda citrifolia (Noni).Journal of Pharmacy and Pharmacology, 59 (12): 1587-1592.


20 Intl. J. Noni Res. 2010, 5 (1-2)

Periyasamy Selvam, Narayanan Murugesh, Myriam witvrouw. 2007. Studies ofcomparative anti-HIV activity and cytotoxicity of Morinda citrifolia L. Noni clinicalResearch Journal, 1 (1-2): 25-27.

Periyasamy Selvam, Christophe pannecuoque. 2008. Studies on anti-HIV andcytotoxicity of leaf extract of Morinda citrifolia L. Noni clinical Research Journal,2 (1-2): 25-28.

Periyasamy Selvam, Christophe pannecuoque, Erik De Clercq. 2009. Studies onanti-HIV activity and cytotoxicity of stem extracts of Morinda citrifolia L. (Noni).Noni clinical Research Journal, 3 (1-2): 47-50.

Philip, S., Rista, S., Dominic, S., Anne, M., James David, V., Jonathan, T.W., Heidi, B.,Susan, K. and Michale, R.B. 1990. New colorimetric cytotoxic assay for anti cancerdrug screening. Journal of National Cancer Institute, 82 : 1107-1112.

Saludes, J., Garson, M.J. and Aguinaldo, A.M. 2002. Antitubercular Constituentsfrom the Hexane Fraction of Morinda citrifolia Linn. (Rubiaceae). PhytotheraphyResearch, 16 (7): 683-8.

Su, B.N., Pawlus AD, Jung HA, Keller WJ, McLaughlin JL and Kinghorn AD. 2005.Chemical Constituents of the Fruits of Morinda citrifolia (Noni) and their AntioxidantActivity. Journal of Natural Product, 68 (4): 592-8.

Umezawa, K. 1992. Isolation of 1-methoxy-2-foremyl-3-hydroxyanthraquinone fromMorinda citrifolia and neoplasm inhibitors containing the same. Japan Kokai TokyoKoho JP 06 87, 736 (94-87, 736) Appl; 92/264, 311 07.

Wang, M.Y., Anderson, G., Nowicki, D. and Jensen, J. 2008a. Hepatic Protection byNoni Fruit Juice against CCl4-Induced Chronic Liver Damage in Female SD Rats.Plant Foods Human Nutrition, (3): 141-47.

Wang, M.Y., Nowicki, D., Anderson, G., Jensen, J. and West, B. 2008b. LiverProtective Effects of Morinda citrifolia (Noni). Plant Foods Human Nutrition, 63(2): 59-63.

Wang MY, West BJ, Jensen CJ, Nowicki D, Su C, Palu AK and Anderson G. 2002Morinda citrifolia (Noni) a literature review and recent advances in Noni research.Acta Pharmacologica Sinica, 23: 1127-41.

West, B.J., Jensen, C.J. and Westendorf, J. 2006. Noni Juice is Not Hepatotoxic. WorldJournal of Gastroenterology, 12 (22): 3616-20.


Intl. J. Noni Res. 2010, 5 (1-2) 21

Antibacterial activities and preliminaryphytochemical analysis of fruit extractsof Noni (Morinda citrifolia L.)

Abstract : Morinda citrifolia L. (Noni) is indigenous to tropical countriesand also used as important traditional folk medicine. Antibacterial activitiesof fruit extracts of M. citrifolia were evaluated against four G+ve and nineG-ve bacteria using disc and well diffusion methods. Preliminaryphytochemical screening of the fruit crude extracts revealed the presencesof flavonoids, saponins, alkaloids, proteins, gums and mucilages, cardiacglycosides, and phenolics. The presence of these bioactive constituents ofthe plant extracts is associated with the antibacterial activities of the plantin both polar and nonpolar extracts viz., petroleum ether, benzene,ethanol, dichloromethane and water extracts. Fruit ethanolic extract showedpromising antibacterial activity against E. coli, S. paratyphi, P. aeruginosa,S. marcescens, V. cholerae, B. subtilis, S. aureus and S. faecalis.Petroleum ether and benzene fruit extracts showed inhibition inhibitoryactivity against both G+ve and G-ve bacteria. Minimum inhibitoryconcentration (MIC) of and minimum bactericidal concentration (MBC)values ranged from 0.03-2.0 mg ml-1 and 0.12-4.0 mg ml-1 respectively.100% of inhibition was recorded at concentration of 2.0-5.0 mg ml-1

against both G+ve and G-ve bacteria. The results obtained with ethanol andpetroleum ether fruit extracts of Noni fruit exhibited significant antibacterialactivity that supports traditional use of the plant for the treatment of somediseases as broad spectrum antibacterial agents.

Key words : Morinda citrifolia, phytochemical, ethanolic extract, antibacterialactivity, MIC, MBC.

Authors’ affiliation :N. Manimala1

S. John Britto1*,L. Joelri Michael Raj1S. Prabhu2

1. The Rapinat Herbarium and Centerfor Molecular Systematics,St. Joseph's College (Autonomous),Tiruchirappalli, Tamil Nadu, India.2. Departmant of Plant Biology andPlant Biotechnology, St. Joseph'sCollege (Autonomous),Tiruchirappalli, Tamil Nadu, India.*Corresponding author:[email protected].

N. Manimala1

S. John Britto1*,L. Joelri Michael Raj1S. Prabhu2

Correspondence to :S. John BrittoThe Rapinat Herbarium and Centerfor Molecular Systematics,St. Joseph's College (Autonomous),Tiruchirappalli, Tamil Nadu, India.

Introduction

Morinda citrifolia L. (Rubiaceae) commonly known as "Indian Mulberry" hasa long history of use as a food in tropical regions of the world (Wang et al.,2002). Noni fruits had been used as food, drink, medicine and colourful dye(Cheeseman, 1903; Maiden, 1889; Merrill, 1943; and Abbott, 1992). The Noniplant is evergreen small tree growing in open coastal regions at sea level andin forest areas up to about 1300 feet above sea level. It's identifiable by itsstraight trunk, large, bright green and elliptical leaves, white tubular flower andits distinctive, ovoid, "grenade-like" yellow fruit. The fruit can grow in size up

22 Intl. J. Noni Res. 2010, 5 (1-2)

N. Manimala et al. Antibacterial activities and preliminary phytochemical analysis offruit extracts of Noni (Morinda citrifolia L.)

to 12 cm or more and has a lumpy surface covered by polygonal-shapedsections. The seeds, which are triangular shaped and reddish brown, have anair sac attached at one end, which make the seeds buoyant (Swanholm et al.,1959).

Noni is reputed to have antibacterial, antiviral antifungal, antitumor,antihelminthic, analgesic, hypotensive, antiinflammatory and immune enhancingeffects (Duke et al., 2002; McClatchey, 2002; Wang et al., 2002; Liu et al.,2001; and Wang and Su, 2001). Allen (1873) reported some information onthe ethno botanical properties of Noni. It's used as herbal medicine withvarious combinations in India, China, Malaysia, Hawaii, Tahiti, Burma, PacificIsland and Australia. The fruit juice is highly demand in alternative medicinefor different kinds of illnesses such as arthritis, diabetes, high blood pressure,muscle aches and pains, menstrual difficulties, headaches, heart disease, AIDS,cancers, gastric ulcers, sprains, mental depression, senility, poor digestion,atherosclerosis, blood vessel problems, and drug addiction (Solomon, 1999).The leaves were applied directly to the skin to treat ulcerations and minorinfections.

Even though ten thousands of antimicrobial compounds exist, the ability ofmicrobes to develop resistance to even the most powerful antimicrobialcompounds is amazing rapid (Gerson et. al., 2006). Center for food andapplied nutrition (FAD) stated that "M. citrifolia has been tested for a numberof biological activities in animal and antimicrobial studies". Some marketingcompanies have proposed that drinking of Noni juice can be used against avery wide variety of ailments. The fruit juice contains a polysaccharide-richsubstance, which has been reported to have antitumor activity (Adesogan,1973; and Hirazumi et al., 1994). The leaf methanolic extract from M. citrifoliawas reported to have antibacterial activity (Nakanishi et al., 1965). In orderto keep pace with this ever increasing need for new antimicrobial, it is imperativethat new compounds be discovered. Scientific information about the therapeuticeffects of Noni on humans is currently limited. The present investigation hasbeen carried out to evaluate the antimicrobial activity and preliminaryphytochemical analysis of in fruit extracts of M. citrifolia which might havepossible application in the treatment of infection diseases.


i. Collection of plant materials

The Noni fruits were collected from the botanical garden of the RapinatHerbarium and Center for Molecular Systematics, St. Joseph's College(Autonomous), Tiruchirappalli, Tamil Nadu, India. (Plate - 1)

Intl. J. Noni Res. 2010, 5 (1-2) 23


ii. Processing of plant material

The collected M. citrifolia fruit was washed with tap water followed by distilledwater. Then fruit was cut into pieces and dried at under shade condition. Thedried fruit was powdered (100-500 µM) with mechanical blender. 50 g of fruitpowder was used to extract using solvents by Soxhlet apparatus sequentially inwith 300 mL of ethanol, benzene, petroleum ether, dichloromethane and water.The process was run for 48 hr after which the sample was concentrated usingrotary evaporated and freeze dried to powdered form. The dried extracts wereweighed and kept in labelled sterile specimen bottles.

iii. Chemicals and solvents

The chemical and solvents which was used in the present study were analyticalgrade and were obtained from Hi Media Laboratories Pvt. Ltd. Mumbai.

iv. Preliminary phytochemical investigations

The phytochemical such as alkaloids, tannins, saponins, terpenoids, steroids,flavanoids, alkaloids, coumarins, quinones, volatile oils and glycosides werescreened according to the methods described by Harborne (1998).

24 Intl. J. Noni Res. 2010, 5 (1-2)


v. Microbial strains

The microbial strains Enterobacter aerogenes; Escherichia coli ATCC 25922;Klebsiella pneumoniae ATCC 15380, Proteus mirabilis; Proteus vulgaris MTCC1771, Pseudomonas aeruginosa ATCC 27853, Salmonella paratyphi , Vibriocholerae, Bacillus cereus, Bacillus subtilis MTCC 441, Serratia marcescens,Staphylococcus aureus ATCC 25923 and Streptococcus faecalis were used inthe present study. The bacterial isolates were cultured in nutrient agar slantsin order to obtain pure isolation and stored at 4°C. Morphological andbiochemical reactions were carried out to ascertain proper identification. Theywere inoculated into nutrient agar slants and stored at 4°C.

Overnight broth cultures of the respective bacterial strains were adjustedto turbidity equivalent to 0.5 McFarland standards (0.2 mL culture of theorganisms was dispensed into 20 mL sterile nutrient broth and incubated for24 h and standardized at 1.50x106 CFU ml-1 by adjusting the optical densityto 0.1 at 600 nm, Tereschuk et al., 1997). For MIC bacterial inoculam wereagitated 1:100 using sterile saline (0.9%) to get concentration of 1.5x108 CFUml-1 respectively.

vi. Agar disc diffusion method

The freeze-dried fruit powder was reconstituted with DMSO to obtain a stocksolution concentrations viz., 100, 50, 25, and 12.5 mg mL-1. Nutrient agarplates were swabbed using sterile cotton swabs with adjusted broth culture ofthe respective bacterial strains. Discs of 6 mm were punched from WhatmanNo.1 filter paper. Up to 10µl of each concentration of the extracts wererespectively introduced in the discs using sterile automatic pipettes. The discswere allowed to dry at room temperature for 2 hrs and were placed atequidistance in each of the plates using a sterile forceps. The plates wereincubated to 37°C for 24 hrs. The control antibiotics Gentamycin (10µg) andKanamycin (30 µg) discs were used as control for G+ve and G-ve bacteriarespectively. The antibacterial activity was expressed as the mean zone of inhibitiondiameters (IZ mm) produced by the plant extract.

vii. Agar well diffusion assay (The agar well diffusion method(Perez et al., 1990; Alade and Irobi, 1993; Abioye et al., 2004)

It is another important method to determine the antibacterial sensitivity of anysubstance (plant extract or antibiotics) on the growth and multiplication of aparticular bacterium. In this method, wells or holes were created using a sterilecork borer on Muller Hinton Agar plates. The inhibition zone formed aroundeach well indicates the antimicrobial activity.

Intl. J. Noni Res. 2010, 5 (1-2) 25


The freeze-dried fruit powder was reconstituted with DMSO to obtain a stocksolution concentration of 200, 100, 50, and 25 mg mL-1. Nutrient agar plateswere swabbed using sterile cotton swabs with the adjusted broth culture of therespective bacterial strains. The plates were incubated to 37°C for 24 hrs.Overnight broth cultures of the respective bacteria strains were adjusted toturbidity equivalent to 0.5 McFarland standards. Five wells (6mm in diameter)were made equidistance in each of the plates using a sterile cork borer. Up to10 µL of each concentration of the fruit extracts were respectively introducedinto the wells using sterile automatic pippettes, with the stock in one well. Theantibiotics Gentamycin (10µg) and Kanamycin (30 µg) discs were used ascontrol for G+ve and G-ve bacteria respectively. The antibacterial activity wasexpressed as the mean zone of inhibition diameters (IZ mm) produced by theplant extract.

viii. MIC and MBC determinations

The tested sample was first of all dissolved in DMSO (stock-200 mg ml-1) andthe solution obtained was added to NBPG (Nutrient broth containing 0.05%phenol red and supplemented with 10% glucose) to a final concentration of4 mg ml-1 (0.1 mL of stock solution to 5 ml of NBPG) for each crudeconcentration within ranges of 0.007 - 4 mg ml-1 for the crude extracts. 100µL of each concentration was added in a well (96-wells micro plate) containing95 µl of NBPG and 5 µl of standard inoculum. The plates were covered witha sterile plate scale, and incubated at 35°C for 24 h. The assay was repeatedtwice. Microbial growth was determined by observing the change of color in thewells (red when there is no growth and yellow when there is growth). Thelowest concentration inhibiting growth of the organisms and is noted andrecorded as the MIC. For the determination of MBC (Minimum Bactericidalconcentration), a portion of liquid (5µl) from each well that showed nochange in color was plated on Nutrient agar and incubated at 37°C for 24 hThe lowest concentration that yielded no growth after this sub culturing wastaken as the MBC. All plates not showing visible growth in the same manner asthe control plate described above are subcultured and incubate at 37oCovernight. Compare the amount of growth from the control plate beforeincubation is compared, which represents the original inoculum. The test mustinclude a second set of the same dilutions inoculated with an organism ofknown sensitivity. These plates are not subcultured the purpose of the controlis to confirm by its MIC that the drug level is correct, whether or not thisorganism is killed is immaterial.

26 Intl. J. Noni Res. 2010, 5 (1-2)


Results and Discussion

Antimicrobial properties of medicinal plants are being increasingly reportedfrom different parts of the world. The world health organization estimates thatplant extract or their active constituents are used as folk medicine in traditionaltherapies of 80% of the words populations. There are about 45,000 plantspecies in India with capacity to produce a large number or organic chemicalsconcentrated hotspot in the region of Eastern Himalayas, of high structuraldiversity.

The qualitative analysis of the ethanol, benzene and aqueous crude fruit extractsshowed the presence of phytochemical such as alkaloids, coumarins, flavonoids,phenolic compounds, saponins, terpenoids and steroids (Table -1). Instead,the phytochemical like anthroquinone and coumarins were absent in petroleumether and dichloromethane extracts. The difference might be due to the natureof solvents which used for extraction determines the presence or absence ofphytochemicals in the extracts. These compounds have potentially significantapplication against human pathogens, including those that cause entericinfections (El-Mahmood et al., 2008). Several authors have linked the presenceof these bioactive compounds to the antimicrobial properties of crude plantextracts (Weimann and Heinrich, 1997; Atindehou et al., 2002; Edeoga et al.,2005; Musyimi et al., 2008; Jayaraman et al., 2008; and Komuraiah et al.,2009). The presence of alkaloids is interesting, as significant quantities areused as antimalarials, analgesics and stimulants (Duke and Ayensu, 1985). Thepresence of glycosides moieties like saponins, phenols and flavonoids whichare also known to inhibit tumor growth and serve also to protect againstgastrointestinal infections are of pharmacognostic importance and give evidenceto the use of the plant in ethnomedicine. Flavonoids on the other hand arewater soluble antioxidants and free radical scavengers, which are capable ofpreventing oxidative cell damage and have strong anticancer activity (Salah etal., 1995; Del- Rio et al., 1997; and Okwu, 2004). Saponins have the propertyof precipitating and coagulating red blood cells (Okwu and Josiah, 2006).Tannins have astringent properties, hasten healing of wounds and inflamedmucous membrane. The qualitative analysis revealed the presence of thebiomolecules such as alkaloids, anthraquinones, coumarins, flavonoids, phenoliccompounds, tannins, saponins, terpenoids and steroids respectively. Acombination of these factors, together with the observed antimicrobial activitymay explain some of the previous claims on the use of the plants in traditionalmedicine. Our results are in agreement with reports that many plant extractshave been as a source of medicinal plants to cure urinary tract infections,gastrointestinal disorders, enhance healing of wounds and stop bleeding, etc.,(Leite et al., 2006; and Okwu and Josiah, 2006).

Intl. J. Noni Res. 2010, 5 (1-2) 27

Table 1 : Phytochemical screening of crude fruits extracts ofMorinda citrifolia L.

S.No. Phytochemical Solvents used

compounds Ethanol Benzene Pet. ether DCM Aqueous

1. Alkaloids + + + + +

2. Terpenoids + + + + +

3. Steroids + + + + +

4. Coumarins + + - + +

5. Tannins + + + + +

6. Flavonoids + + + + +

7. Phenols + + + + +

8. Volatile oils + + + + +

9. Anthraquinones + + + - +

10. Saponins + + + + +

(+)= Detected; (-) = Not detected. Values are mean of triplicate determinationson dry weight basis.

Agar disc diffusion method

The evaluation of the activity of ethanol, petroleum ether, benzene, DCM andaqueous extract of fruits of M. citrifolia using the disc diffusion method is givenin (Tables -2). The results were observed in terms of inhibitory zone aroundeach disc caused by diffusion of antibacterial properties from the plant extractimpregnated disc into the surrounding medium. As can be seen from tables,various solvent extracts reveal varied effectiveness. The ethanol and petroleumether extracts were found to be inactive against all the 13 organisms tested.(Plate - 2)


28 Intl. J. Noni Res. 2010, 5 (1-2)

Table- 2 : Antibacterial activity of fruit extracts of Morindacitrifolia L. by agar disc diffusion method

E. a - Enterobacter aerogenes; E.coli - Escherichia coli ; K. p - Klebsiellapneumoniae; P. m -Proteus mirabilis; P. v - Proteus vulgaris; P. a -Pseudomonas aeruginosa; S. p - Salmonella paratyphi; S. m -Serratiamarcescens ; V. c - Vibrio cholerae; B.c - Bacillus cereus; B.s - Bacillussubtilis; S.a - Staphylococcus aureus; S.f - Streptococcus faecalis. Eachvalue represents the mean of triplicate analysis. Standard deviation was 0.05 to0.10 for the values.


Gram Negative Bacteria Gram Positive Bacteria

Intl. J. Noni Res. 2010, 5 (1-2) 29


The ethanolic extracts exhibited high degree of inhibition followed by Petroleumether, Benzene, DCM and aqueous. Among microorganism the extracts at theconcentration of 1000 µg and 500 µg of gram negative bacteria showed highestrange of inhibition when compared with gram positive bacteria. The fact thatthe extract produced inhibitory activities comparable to the reference drugampicillin on all the tested microbes including S. paratyphi (19.4mm) andV. cholerae (19.0mm) is indicative of highly potent antibacterial constituentsespecially of antityphoid activity for which this plant is used in folklore medicine.P. aeruginosa (18.9mm), E.coli (18.4mm), and S. marcescens (18.1mm)which is already known to be multi-resistant to drugs, was also resistant to theplant extracts tested. It was susceptible only to benzoic acid and cinnamic acid.Then the other gram negative organism also had moderate activity againstK. pneumoniae (17.5mm), E. aerogenes (15.2mm), P. vulgaris (14.5mm),P. mirabilis (14.2mm) respectively. When compared with gram negative bacteriathe gram positive bacteria with moderate inhibition zone was recorded inS. aureus (17.6mm), S. faecalis (16.6mm), B. subtilis (16.3mm) andB. cereus (15.1mm) which is also resistant to different antibiotics. This moderateantimicrobial activity of gram positive bacteria may be attributed to the reducedability of these solvents to extract phytochemical compounds or the presenceof pigments or phenols, which are known to interfere with antimicrobial activity(Doughari, 2006). Extracts of 1000µg, 500µg, showed wider range of inhibitionwhile 250 µg and 125 µg had minimum or negligible inhibition.

The petroleum ether extract of 1000 µg and 500 µg for fruit extract had widerzone of inhibition against S. paratyphi (17.5mm), V. Cholerae (16.8mm), P.aeruginosa (12.5mm), S. marcescens (15.8mm) and E. coli (15.6mm)respectively while others organisms showed low activity. The concentration of500 µg and 250µg showed moderate activity against the rest of the organismstested for antibacterial activity when compared to standard antibiotics.

The Benzene, DCM and aqueous extract showed moderate inhibition against allthe test bacteria. This might have resulted from the lack of solubility of theactive constituents in aqueous solutions. The diameter of inhibition zones foreach of the samples were compared with standard antibiotic. It was noted thatthe inhibition zones of the samples to be either less than or greater than orequal to the inhibition zones of standard antibiotics and also antimicrobialstudies were done in triplicates and diameters of zones of inhibition (mm) areexpressed as the mean and standard errors. (SEM).

Agar well diffusion method

Antibacterial activity of crude extracts of M. citrifolia fruits were evaluated bymeasuring the diameters of zones of growth inhibition on some members of the

30 Intl. J. Noni Res. 2010, 5 (1-2)


Enterobacteriaceae and the results are presented as shown in Table 3. All thetest organisms were susceptible to Morinda fruit extracts though to varyingdegrees. Karou et al. (2006) reported that the susceptibility of bacteria to plantextracts, on the basis of inhibition zone diameters varied according to strainsand species which also is similar to the data obtained in this study. The highestzone of growth inhibition was shown by ethanol extract (1000µg) against Gramnegative bacteria S. paratyphi (18.4mm), V. cholerae (18.0mm), S. marcescens(17.9mm), E. coli (17.5mm) and P. aeruginosa (17.3mm). The growthinhibition was moderate against gram positive bacteria S. aureus (16.2mm),B. cereus (15.6mm), B. subtilis (15.1mm) and S. faecalis (14.9mm). At thesame concentration (1000 µg) ethanol extract also inhibited P. vulgaris(16.2mm), K. pneumoniae (15.5mm), P. mirabilis (15.4mm) and E.aerogenes (14.3mm) and they show considerable activity against gram negativebacteria than gram positive bacteria. These results show that gram negativebacteria are resistant to the action of tested extracts and the same have beenreported by several researches for other plant based extracts (Locher et al.,1995; Wang, 2002; Duduku 2007). The reason for the differential sensitivitybetween gram positive pattern and gram negative bacterial strains could not beascribed to their morphological differences or adduced to their chemicalcompositions. Gram negative bacterial strains have an outer phospholipidsmembrane with the structural lipopolysaccharide components, which maketheir cell wall impermeable to antimicrobial agents (Nikaido and Vaara, 1985).

Table-3 : Antibacterial activity of fruit extracts of Morindacitrifolia L. by agar well diffusion method

Intl. J. Noni Res. 2010, 5 (1-2) 31


E. a - Enterobacter aerogenes; E.coli - Escherichia coli ; K. p - Klebsiellapneumoniae; P. m -Proteus mirabilis; P. v - Proteus vulgaris; P. a -Pseudomonas aeruginosa; S. p - Salmonella paratyphi; S. m -Serratiamarcescens ; V. c - Vibrio cholerae; B.c - Bacillus cereus; B.s - Bacillussubtilis; S.a - Staphylococcus aureus; S.f - Streptococcus faecalis. Eachvalue represents the mean of triplicate analysis. Standard deviation was 0.05 to0.10 for the values.

MIC and MBC determinations

MIC test of Noni fruit was done selectively against eight organisms that werefound susceptible in agar diffusion assay. They are Escherichia coli, Vi. cholerae,P. aeruginosa, S. paratyphi, B. cereus, B. subtilis, S. aureus and S. faecalis.MIC values also showed that extracts were able to inhibit both gram positivebacteria and gram negative bacteria at lower concentrations, The lowest MICfor Morinda citrifolia fruit ethanol extract was recorded against S. paratyphi,(0.03mg/ml), Vibrio cholerae (0.03mg/ml), P. aeruginosa (0.06mg/ml), E.coli (0.12mg/ml), S. aureus (0.25mg/ml), B. cereus (0.25mg/ml) and S. faecalis(0.5mg/ml) as detailed in Table -4. The ethanol extract gave an MBC value forS. paratyphi (0.12mg/ml), V. cholera (0.12mg/ml), P. aeruginosa (0.12mg/ml), and E. coli ((0.12mg/ml) respectively, while for and B. subtilis, B. cereusS. faecalis and S. aureus the MBC values were 0.5-1 mg/ml, respectively(Table- 5). Petroleum ether extract gave an MBC value for S. paratyphi (0.5mg/ml), V. cholerae (0.5mg/ml), P. aeruginosa (0.5mg/ml), and E. coli ((1mg/ml) respectively, while for and B. subtilis, B. cereus S. faecalis and S. aureusthe MBC values were 1-4 mg/ml, respectively. For Benzene, Dichloromethaneand Aqueous extract, the MBC values were Resistant (R) for all the pathogens.Agents with low activity against a particular organism usually gives high MIC andMBC values, while a highly reactive agent gives low MIC and MBC values. TheMIC and MBC techniques are used to evaluate the efficacies of antimicrobialagents and in this study, the MIC and MBC values tend to support the resultsobtained in the antibacterial screening by agar diffusion showing clearly thatthe ethanol extracts were more potent than either Petroleum ether, Benzene,Dichloromethane, and Aqueous extracts. Hugo and Russell, (1984) havereported that the MBC values can either be the same or higher than the MIC

Gram Negative Bacteria Gram Positive Bacteria

32 Intl. J. Noni Res. 2010, 5 (1-2)


values. In this study, the MIC values were either the same or slightly lower thanthe MBC values, similar to the results of Karou et al. (2006). The MIC and MBCvalues are predictive of the efficacy of agents' in-vivo. However, the MBC valueswhich are obtained after plating various dilutions of the extracts, is morereliable than the MIC values (Junaid et al., 2006). (Table 4; 5). (Plate - 3)

Intl. J. Noni Res. 2010, 5 (1-2) 33

Plate - 2Antibacterial activity of fruit extracts of

Morinda citrifolia L. by agar disc diffusion method

Salmonella paratyphi Serratia marcescens

Pseudomonas aeruginosa Vibrio Cholerate

Bacillus cereus Staphylococcus aureus


Table 4 : Minimum Inhibitory Concentration (MIC) of fruits inMorinda citrifolia L.

MIC values < 0.5 mg/ml (500 µg /ml) were considered to be active, Antibiotic-C1- Kanamycin 30 mg/ml, C2- Gentamycin 10mg/ml.

Table 5 : Minimum Bactericidal Concentration of fruits inMorinda citrifolia L.

Resistance (R) MBC values < 0.5 mg/ml (500 µg /ml) were considered to beactive

Conclusion

Acubin, L-asperuloside and alizarin in the Noni fruit, as well as some otheranthraquinone compounds in Noni roots are all proven antibacterial agents.These compounds have been shown to fight against infectious bacteria strains

34 Intl. J. Noni Res. 2010, 5 (1-2)


such as P. aeruginosa, Proteus morgaii, Staphylococcus aureus, Bacillussubtilis, Escherichia coli, Salmonella and Shigella. These bactericidalcompounds found in Noni are responsible for the treatment of skin infections,colds, fevers, and other bacterial-caused health problems. Bushnell (1950)reported on the antibacterial properties of some plants found in Hawaii,including Noni. He further reported that Noni was traditionally used to treatbroken bones, deep cuts, bruises, sores and wounds. Extracts from the ripeNoni fruits inhibited Pseudomonas aeruginosa, M. pyrogenes, and E. coli andwere also shown to have moderate antibacterial properties against Salmonellatyphosa, Salmonella montevideo, Salmonella schottmuelleri, and Shigellaparadys. Lovett (1986) demonstrated that ethanol and Methanol extractsobtained from Cycas circinalis, Morinda citrifolia, Bridelia penangiana,Tridax procumbens, Hibiscus tiliaceus and Hypericum papuam showedantibacterial activity. The widespread medicinal use of these plants would suggestthat they do contain pharmacologically active substances and alternative methodsof extraction and screening should be utilized to find the major bio-activecomponent in the plants for the purpose of new drug development. Smith(1998) reported that selected plants including M. citrifolia have a history ofuse in Polynesian traditional medicine for the treatments of infectious disease.

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Liu, G., Bode, A., Ma, W.Y., Sang, S., Ho, C.T. and Dong, Z. 2001. Two novelglycosides from the fruits of Morinda citrifolia (Noni) inhibit Ap-1transactivation and cell transformation in he mouse epidermal JB6 cell line.Cancer Research, 61(150): 5749-56.

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38 Intl. J. Noni Res. 2010, 5 (1-2)

Noni as an intercrop in Coconut

Abstract : Coconut is a wide spaced perennial crop remained committedto land for more than 65 or 75 years. This provides ample scope for inter/ mixed crop at various stages. Noni as a mixed crop in coconut is comingup well. Since it is only four years time, the performance has to be stillstudied.

Key words : Noni, Intercrop, Coconut

George V. Thomas

Authors’ affiliation :George V. ThomasDirectorCentral Plantation Crops ResearchInstitute, [email protected]@gmail.com

Correspondence to :George V. ThomasDirectorCentral Plantation Crops ResearchInstitute, [email protected]@gmail.com

Introduction

Coconut is a traditional plantation crop of India, assumed the status of an importantcommercial crop with an annual production of 15729.7 million nuts (2008-09)(Anon., 2010). In India, most of the acreage under coconut palm (90%) lies in thefour southern states i.e., Kerala, Tamil Nadu, Karnataka, and Andhra Pradesh. Keralaranks first in area (53.76%) and production (45%) followed by Tamil Nadu,Karnataka and Andhra Pradesh. Coconut is mostly a crop of small holders in India,the average size of a holding being 0.22 ha. More than 90 % of the five millioncoconut holdings in the country are less than one ha in size (Thampan, 1988). Theincome derived from such small holdings is not sufficient to sustain even the smallfamilies. Under monocropping of coconut, the palm yields are poor and farmersare underemployed with only 100 and 120 man-days of employment under rainfedand irrigated systems respectively. Another problem associated with coconut industryis that, a major portion of the produce is locally consumed and less importance isgiven to product diversification. Coconut groves offer excellent opportunities toexploit the inter-space potential in the garden for maximizing returns per unit areaand improving the employment generation. Coconut based cropping/farming systems,involving cultivation of compatible crops in the interspaces of coconut and integrationwith other enterprises like dairying offer considerable scope for increasing productionand productivity per unit area, time and inputs, by more efficient utilization ofresources like sunlight, soil, water and labour. In humid tropics, higher efficiencyof utilisation of the basic resources of crop production viz. land, solar radiation andwater can be achieved by adopting intensive cropping systems (Nelliat, 1973).

Intl. J. Noni Res. 2010, 5 (1-2) 39

George V. Thomas, Noni as an intercrop in Coconut

Coconut based cropping/farming system

A system aims at crop diversification and intensive cropping in the interspaceavailable in the coconut to increase the per palm productivity as well as productivityof unit holding in a system approach in which the available farm resources like soiland water/rainfall resource, farm labor, agricultural inputs (seeds, fertilizers, agro-chemicals) are utilized to produce both food and non-food agricultural productsfrom the farm, in a profitable way. In coconut based cropping/farming system, allmanagement practices and component production systems should be able to maintainthe high productivity, profitability and sustainability of the existing stands of thecoconut crop to maximize the economic yield of the farm. Sustainability is the mainobjectivity of farming system where production process is optimized through efficientutilization of the inputs in safeguarding the environment.

Scope and opportunities of growing intercrops in coconut

Resource use in coconut

Coconut being a perennial crop, is committed to land for sixty to hundred years.Since the maximum required horizontal and vertical space cannot be altered duringgrowing period, a wider spacing is provided from the time of field planting wherebycoconut alone does not utilize the air and soil space fully in the beginning and lateryears. Crown shape and length of coconut leaf necessitate a wider spacing of 7.5x 7.5 m giving a plant population of about 175 palms/ hectare. However, experimentalevidences show that a sole crop of coconut, at the recommended spacing of 7.5x 7.5 m does not fully utilize the available basic resources of crop production,namely, soil and solar energy water and nutrients.

Rooting pattern of Coconut

Coconut palm like all monocots has a typical adventitious root system. Underfavorable conditions, as many as 4000 to 7000 roots are found in the middle agedpalms. Kushwah et al. (1973) reported that about 74 % of the roots produced bya palm under good management did not go beyond 2 m lateral distance and 82% of the roots were confined to the 31 to 120 cm depth of soil. Anil Kumar andWahid (1988) observed that more than 80 % of the root activity was confined toa lateral distance of 2m from the trunk. Further root studies on adult palm grownunder coastal sandy soil confirmed that the mean number of roots produced were5072. Over 95.5 % of the roots were found in the top 0-120 cm depth. Top 0-30cm depth had 18.9 % of roots and 63 % of roots were confined to 31-90 cm depth.With respect to lateral spread maximum % of the roots emerging from the bole wereconfined to 2m radius and only 12.2 and 7.5 % of roots were extended up to 3mand 4m radius respectively (Maheswarappa et al., 2000). Thus the active root zone

40 Intl. J. Noni Res. 2010, 5 (1-2)


of coconut is confined to 25 % of the available land area and the remaining areacould be profitably exploited for raising subsidiary crops. By growing a suitableintercrop with different root depth and volume in the coconut garden, the appliedand the native nutrients can be mined from different layers of soil both horizontallyand vertically whereby the nutrient use efficiency can be improved in the coconutbased cropping system. Thus a high efficiency in the use of available soil moistureand nutrients can be achieved by growing intercrops outside a 2 m radius aroundthe base of the palm.

Canopy structure and light utilization

The Venetian structure of the coconut crown and the orientation of leaves allow partof the incident solar radiation to pass through the canopy and fall on the ground.In the initial years of planting, the leaf size is small and the orientation of leaf is invertical angle with slight slanting position but as palm grows the size of the leavesincrease fast and almost attain the full size in the fifth or sixth year of planting.However the orientation is still more towards vertical angle. The variety, soil type,nutrient status and the management practices play a greater role in the leaf size. Thelight intensity at ground level was always higher than 6700 lux at all the periods ofthe year ( Nair, 1979) The leaves in a coconut palm crown are not randomlydistributed, but clumped around a few widely spaced growing points. This non-random distribution will also lead to low extinction coefficient of around 0.65 forPAR. Age and spacing of coconut palms, soil fertility, varietal characteristics, leaf areaand time of the day influence the light penetration through the canopy. With themovement of the earth and the movement of coconut fronds in the wind, the lightand shade patterns under the palms are constantly changing. The distribution oflight at different positions in the canopy zone of coconut varies very much becauseof the non-random distribution of leaves. This causes differences in the growth andyield of intercrops at different positions of the plantation floor.

The amount of light transmitted ranges from 5 % in a five to ten year old D x T hybridat a density of 560 palms/ha to about 90 % in a 60-70 year old plantation at a densityof 120 palms/ha. Nair and Balakrishnan (1976) estimated that as much as 56%of the sunlight was transmitted through the canopy during the peak hours (10-16hours) in palms aged around 25 years. The diffused sunlight facilitates growing anumber of shade tolerant crops in the interspaces. The apparent coverage ofground and shade cast by the canopy, and the magnitude of radiation transmittedthrough the canopy vary according to the age of palms as shown in Fig..1 (Nelliatet al., 1974).

Intl. J. Noni Res. 2010, 5 (1-2) 41


Based on the growth habit of the palm and the amount of light transmitted throughits canopy, the life span of coconut palm could be divided into three distinct phasesfrom the point of view of intercropping.

1. Planting till full development of canopy (about 5- 8 years): A major portion ofthe solar energy is not being intercepted by coconut leaves, good transmissioninitially, as the palm canopy develops with age, the percentage of utilisation increasesprogressively during the pre-bearing period, with a corresponding decrease intransmission of light suitable for growing annuals/biennials. The suitable crops arecereals, grain legumes, vegetables, spice crops, fruit crops like banana, pineappleetc.

2. Young palms (8 to 20 years): Maximum ground coverage and high canopy size,moderate to poor light availability- not congenial for multiple cropping.

3. Mature trees (more than 20 years): Increase in trunk height; reduction in crownsize light transmission increase with age (high light levels) - the amount of slant raysof sun falling on the ground increases and consequently, the apparent coverageof ground by the canopy of coconut decreases progressively ideal for raising annualand / or perennial crops as multiple and multi-storeyed cropping models. Thesuitable intercrops are banana, pineapple, black pepper, beverage crops like cocoa,coffee, spice trees like nutmeg, clove and fruit crops like rambutan, mangosteen,citrus etc.

Criteria for selection of subsidiary crops

1. Crops should be selected according to their shade tolerance and amount ofsolar radiation available.

42 Intl. J. Noni Res. 2010, 5 (1-2)


2. Should not grow as tall as coconut.

3. Should not be more susceptible than the main crop to diseases they have incommon.

4. Should not require harvesting or other operations which would damage themain crop or induce soil erosion or damage soil structure.

5. Should not have an economic life longer than the main crop.

6. Its root system should exploit different soil horizons/zones.

7. Crops should be selected according to the soil type, rainfall pattern/irrigationfacilities and climatic conditions.

8. Availability of marketing/processing facilities and labour availability.

It is not very necessary always, to fulfill all these requirements to have successfulcrop combinations. But one must ensure that the correct crop is chosen. Inmost cases failure of inter/mixed cropping is due to the wrong choice of thecrop(s).

Relevance of Coconut Based Cropping Systems (CBCS) in India

The practice of CBCS can provide:

1. Food security through food sufficiency;

2. Nutritional foods rich in vitamins and minerals (nutrients);

3. Employment generation from farm diversification; and

4. Ecological stability (environmental protection).


Noni as intercrop

Morinda citrifolia L., known commercially as Noni, grows widely throughout thePacific and is one of the most significant sources of traditional medicines amongPacific island societies. This small evergreen tree or shrub is native from SoutheasternAsia (Indonesia) to Australia, and now has a pantropical distribution. Noni is notedfor its extremely wide range of environmental tolerances. It grows in infertile, acidic,and alkaline soils. It is found naturally in relatively dry to mesic sites or lowland areasin close proximity to shorelines, or as an important forest understoried species inlow-elevation Pacific island forests and rainforests. All parts of the plant have traditionaland/or modern uses, including roots and bark (dyes, medicine), trunks (?rewood,tools), and leaves and fruits (food, medicine). The medicinal applications, bothtraditional and modern, span a vast array of conditions and illnesses, although most

Intl. J. Noni Res. 2010, 5 (1-2) 43


of these have yet to be scienti?cally supported. Noni is well suited for intercroppingwithin traditional agroforestry subsistence farming systems or as a monocrop in fullsun. The tree has attained signicant economic importance worldwide in recent yearsthrough a variety of health and cosmetic products made from its leaves and fruits.These include fruit juices as well as powders from the fruit or leaves. Noni isrelatively easy to propagate from seeds, stem cuttings, and Tissue culture. Thepreferred methods of propagation are by seed and by cuttings from stem verticals.Intercropping systems include traditional subsistence farming of intercropping withbreadfruit, kava, papaya, mango, coconut, banana, timber species, coastal shrubs,and grasses while in modern commercial intercropping Noni is grown with papayaand coconut as single hedge/double hedge.

Results and Discussions

The experiment conducted at Central Plantation Crops Research Institute, Kasaragodrevealed the following results. The growth attributes such as plant height, totalnumber of branches, girth size and canopy spread of different planting materialsplanted as single hedge and double hedge was observed after one and half year offield planting.

In general, the tissue cultured plant's performance was poor in the sandy soil of westcoast region. This may be because the establishment itself was poor. Due to poorperformance and late pick up in growth the plant height, total number of branches,girth size and canopy spread were less compared to the materials raised from seedsand cuttings (Table 1). Between single hedge and double hedge, marginal differenceswere noticed in all the growth parameters showing double hedge performing bettercompared to single hedge planted in tissue culture plants. However it is too earlyto draw any conclusion. The seedlings raised from seeds brought from NRC onNoni, Chennai also suffered for establishment when field planted as they are notnative to Kasaragod. There are a few gaps in this category; however it is better thanthe tissue culture plants in establishment.

Experimental details

1. Three types of planting materials viz seedling raised through seeds, stem, cuttingsand tissue culture plants.

2. Single and double hedge system of Noni in Coconut in adopted.

44 Intl. J. Noni Res. 2010, 5 (1-2)


Table 1. Growth attributes of Noni grown as intercrop in coconut

Treatment Plant Total Girth Canopy spreadheight No. of size (cm)(cm) branches (cm) East - North -

West South

Single hedge (SH)

Tissue culture 99.8 8 9.2 104 102

Seedlings 167.0 13 13.3 160 137

Cuttings 156.8 16 14.2 204 189

Double Hedge (DH)

Tissue culture 128.6 9 10.0 91 111

Seedlings 151.2 15 12.6 163 170

Cuttings 144.6 17 13.0 179 183

Between the double hedge and single hedge plantings, there was no clear cutdifference in different growth parameters. Stem cuttings (5 to 10cm thickness and20-30cm length) were chosen for raising seedlings. It was noticed that thicker stemspicked up faster and may be due to more stored food materials in the thicker stem.These rooted cuttings were planted in the field along with tissue culture seedlingsand stem cuttings. The establishment of stem cutting in the field was very good andthere was no mortality. In this category, there are indication that single hedgeperforms better than double hedge. Thus in two years, there is no markabledifference observed in growth parameters.

Yield of Noni in coconut garden

Among the different planting materials (Table 2) it was observed that flowering andfruiting were early in the rooted cuttings which are native of Kasaragod (thecuttings were collected from the naturally grown Noni from the Beach side, andseedlings and the tissue culture plants collected from the World Noni ResearchFoundation, Chennai). Among the different planting materials tried, the cuttings gavemore number of fruits as they established early supported with better growthparameters. The fruit weight is markably less than the tissue culture plants andseedlings. The fruit weight of the seedlings and number of fruits in the seedlingsare second in order compared to the cuttings , mainly due to establishment and

Intl. J. Noni Res. 2010, 5 (1-2) 45


acclimatization problem in the beginning. However, the fruit weight to be better. Thetissue culture plant weight is

Table 2. Yield of Noni grown as intercrop in coconut

Treatment Total Fruit Yield (No) Average Fruit weight (g)

Single hedge

Tissue culture 16 33.0

Seedlings 65 25.0

Cuttings 378 17.5

Double Hedge

Tissue culture 98 25.9

Seedlings 140 21.5

Cuttings 740 16.5

more than fruit weight of the seedlings and cuttings. Number of fruits in the categoryis less due to poor performance both in establishment and acclimatization. It istoo early to present any confirmed result.

Fig 1. Noni intercrop in coconut garden

Yield of Coconut in Noni intercropping system

Coconut responds well to water and nutrient supply and is clearly indicated in yieldperformance of the coconut in Table 3. There is an overall increment in yield incoconut. Introduction of any intercrop in coconut and supply of water and nutrientto the inter crop incidentally will improve the coconut yield , experienced in manystudies at CPCRI,Kasargod. Though, 80% of coconut roots are restricted to the basinarea of 2m radius of the coconut. There are active roots beyond the 2 m radiusand Noni in the inter-space of coconut also competes for nutrition. Thus the inputssupplied to the intercrops are also shared by the main crop in production of higheryield. Among the different planting materials and planting methods, no markabledifference was observed in the coconut yield. However, yield increment (Fig 2) isnoticed in the main crop coconut to the tune of 22 to 26% in the single hedge and

46 Intl. J. Noni Res. 2010, 5 (1-2)


double hedge intercrop compared to the actual nut yield after two years of startingof the experiment.

Table 3. Yield of coconut in Noni intercropping system

Trees Single Hedge Double hedgePre treatment Post treatment Pre treatment Post treatment

1. 22 42 23 25

2. 50 57 61 70

3. 51 65 55 77

4. 28 51 60 68

5. 96 106 32 42

6. 25 37 62 82

7. 22 25 60 53

8. 55 68 27 48

Total 357 451 380 465

Fig 3. Percent yield increase in coconut when Noni grown asintercrop

Conclusion

From the result of this study of two years it is clearly seen that, it is too preliminaryand continuing the study for more years may yield some result which will be usefulfor higher density to get more yield from unit area. However, it is high time toconduct research in Noni as a intercrop in coconut garden as day by day people

Intl. J. Noni Res. 2010, 5 (1-2) 47


are more health conscious. No doubt in India, Noni is gaining importance fastbecause of its traditional and/or modern uses, including roots and bark (dyes,medicine), trunk (fire wood and tools) and leaves and fruits (food and medicine).The medicinal application has a vast array of condition and illness, although mostof these have yet to be scientifically supported. A good support for Noni cultivationis possible only when there is a tie between the growers and companies through abuy back policy with supply of good quality planting materials.

References

Anonymous. 2010. Directorate of statistics and economics. Ministry of Agriculture,GOI, New Delhi.

Anil Kumar, K.S. and Wahid, P.A. 1988. Root activity pattern of coconut palm.Oleagineux 43:337-340.

Hawaii Plant Conservation Center. 1992. Noni. National Tropical Botanical Garden,Kauai. 2 pp.

Kushwah, B.L., Nelliat, E.V., Markose, V.T. and Sunny, A.F. 1973. Rooting pattern ofcoconut. Indian J. Agron., 18:71-74.

Maheswarappa, H. P., Subramanian, P. and Dhanapal, R. 2000. Root distributionpattern of coconut (Cocos nucifera L.) in littoral sandy soil. J. Plantn. Crops., 28(2): 164-166.

Morton, J. 1992. The ocean-going Noni, or Indian mulberry (Morinda citrifolia,Rubiaceae) and some of its colourful relatives. Economic Botany, 46: 241-256

Nair, P.K.R, and Balakrishnan, T.K. 1976. Pattern of light interception by canopiesin coconut-cocoa crop combination. Indian J. Agic. Sci., 46:453-462.

Nair, P.K.R.1979. Intensive multiple cropping with coconut in India, Principles-programmes-Prospects. Verlag Paul parcy- Berlin and hamburg. Adv. In Agron andCrop sci. 6.147 p (ICRAF, Nairobi, Kenya)

Nelliat, E. V. 1973. Multiple cropping or multi-storeyed cropping in plantation crops.Plant. Crops, 1 (Suppl): 204.

Nelliat, E. V., Bavappa, K. V. A. and Nair, P. K. R., 1974. Multi-storeyed cropping -A new dimension in multiple cropping for coconut plantations. World Crops, 26 (6): 262-266.

Nelson, S.C. 2001. Noni cultivation in Hawaii.University of Hawaii. CTAHR -cooperative extension service. PD-19.

Thampan, P.K. 1988. The coconut profile of India. In: Status of coconut researchand Development in India, Malaysia, Srilanka and Tanzania., pp 1-22, Kerala Ag.Univ. Vellanikkara, Kerala, India

48 Intl. J. Noni Res. 2010, 5 (1-2)

Insect diversity on Morinda citrifolia L.in west coast of Kerala

Keywords : Natural vegetation, green hopper, beneficial insects, west coast

S.P. Shanthakumar,*S. Malarvannan,V. R. PrabavathySudha Nair

Authors’ affiliation :S.P. Shanthakumar,*S. Malarvannan,V. R. PrabavathySudha NairM. S. Swaminathan ResearchFoundation, III Cross Street,Taramani Institutional Area,Chennai -600 113Email: spshanthakumar@ gmail.com

Correspondence to :S.P. ShanthakumarM. S. Swaminathan ResearchFoundation, III Cross Street,Taramani Institutional Area,Chennai -600 113Email: spshanthakumar@ gmail.com

Abstract : The canopy of natural vegetation is believed to be the key to theimmense diversity of insects. Natural vegetation of Morinda citrifolia L. wassurveyed in six coastal districts of Kerala for insect diversity. From the survey,the major insect pests associated in the Morinda habitat recorded were Bemesiatabaci, Siphanta acuta and Flata ocellata, Halyomorpha halys, Macroglossumgyrans, Leptocentrus tarus, Orthacris maindroni, Nezara graminea, Drosophilamelanogaster, Aleurocanthus woglumi, Henosepilachna vigintioctopunctata,Psara obscuralis, Technomyrmex albipes, Maconellicoccus hirsutus andCoccus viridis. The beneficial insects observed were Oecophylla smaragdina,Alcaeorrhynchus grandis, Chilomenes sexmaculatus, Xylocopa varipuncta,Camponotus compressus, Anegleis cardoni, Myrmicaria brunnea, Apis dorsataand Cryptolaemus montrouzieri. The sampling techniques involved data analysison taxonomic and guild structure, species abundance and biomass of insectsand the species similarity among six districts of the west coast of Kerala.

Introduction

Morinda citrifolia is a tropical evergreen shrub also known as Indian mulberry,naturally distributed along the west coast line of India predominantly in States suchas Kerala and Karnataka. It is rich in macro and micronutrient with strong historyof medical application. Noni had been used as a food, drink, medicine and dye(Abbott, 1992). It has been reported to have a broad range of health benefits forcancer, infection, arthritis, diabetes, asthma, hypertension and pain (Whistler, 1992).In the process of conservation Morinda spp it is necessary to establish a record onthe major associated fauna which are different groups of insects is a very importantfunctional entity, and thus their abundance and species richness is critical to balanceand maintain the diversity of terrestrial ecological systems everywhere (Thomas etal., 2008). Knowledge of the insect- pest complex associated with any crop isessential for developing pest control strategies that leads to less crop damage andeconomic loss. In this context, it is felt important to study the diversity, distributionand insect-plant interaction in Kerala where the natural occurrence of M.citrifoliais abundant. The objective of the study is to conduct a survey and inventory on the

Intl. J. Noni Res. 2010, 5 (1-2) 49

diverse community of insects and their association with the natural vegetation ofMorinda citrifolia in Kerala district. This will provide information for future researchand crop protection.


A preliminary survey on the natural distribution of M.citrifolia was conducted alongthe west coast of Kerala. Based upon the distribution pattern this study focused onthe six coastal districts, located at approximately (12°23?33.26 N, 75°01?50.87 E,)along the western coastline of the Kasargod to Alleppey (9°29?13.93 N, 76°19?09.05E,) Kerala, India (Table 1). The area surveyed falls under six districts viz., Kasargod,Kannur, Kozhikode, Thrissur, Ernakulam and Alleppey with elevations or tidal rangefrom 10m-1 m. The vegetative community in these geographical locations is discrete.Plant community structure in this area is varied and range from open grasslands withscattered shrubs and trees, dense thick, to gallery forests along the major rivermouths. To sample this large geographic area within a reasonable amount of time,linear sampling transects based on distance and distribution was established alongthe major coast lines, as transect Z walk sampling is not feasible in long linedistribution. Sampling was done in M.citrifolia which was of different ages,approximately 3 -9 m tall plants during the investigation. Flowering and Non floweringstages were sparsely distributed across the landscape. During the observation period,insects were collected from foliage, inflorescence, fruit and stem using standardaerial insect nets and plastic insect vials manually. Smaller insects were collected byshaking the flower into nets by gentle tapping as well as aspirators. Insects wereimmediately anesthetized and put on collection containers for study in the laboratory.Data collected from each plant included latitude / longitude coordinates, numberof insect visitors and number of insect morphospecies. Basic summary statistics(average number of visitors per plant, average number of insect visitors per plant,average number of insect species per plant) were calculated for each plant species.

Results and Discussion

Data assessing species composition of insect communities and presence of herbivoresas well as natural enemies were collected across the diversity gradient of samplingsites were analysed. (Fig. 1) The results revealed that mealy bugs, Sphingid caterpillars(Foliage feeders), white fly, leaf hopper, weaver ants (Fig. 2) was abundant in all sixdistricts taken for the study (Table 2). It was followed by sucking pests like aphidsand other insects like lace bugs, thrips and loopers. A meager incidence of leafminer was also noticed. Among the natural enemies, spiders and coccinellid beetleswere present.

S.P. Shanthakumar et al. Insect diversity on Morinda citrifolia L. in west coast of Kerala

50 Intl. J. Noni Res. 2010, 5 (1-2)

Table 1. Geographic distribution of M. citrifolia and sampling sites at Kerala

Table 2 : Pest status on M. citrifolia in West coast of Kerala


Intl. J. Noni Res. 2010, 5 (1-2) 51

Table 3: List of insects indentified on M.citrifolia in West coast of Kerala

Fig. 1. Abundance of insect pest and natural enemies in natural stands ofM. citrifolia in west coast line of Kerala


52 Intl. J. Noni Res. 2010, 5 (1-2)

Fig. 2. Major insects associated on M. citrifolia1. Macroglossum gyrans; 2. Psara obscuralis; 3. Oecophylla smaragdina;

4. Bemisia tabaci; 5. Cheilomenes sexmaculatus

In Alleppey district, nine places namely, Kinakari, Meenapalli lake, Pathiramanal andplains were surveyed. Similar trend of more Eriophyid mites was present in Alleppeybeach, Tottampalli, Totankulangara, Vembanad, Pamba canal, as in the case ofKasargod and Kannur. In addition, in this district the other dominant pests werewhite fly and ants (Table 2). The defenders recorded were spiders and Coccinellidbeetles. However, the survey conducted at Kinakari, Meenapalli Lake, Pathiramanaland plains of Allepey showed a different trend. In these places the Eriophyid mitewas absent while, leaf hopper, looper and white fly was predominant (Table 2).

Sphingid caterpillars were observed on plants present in Kasargod, Kannur andAllepey. This was on par with Stampsfiji.com (2002) which reported that Noni is theprincipal host of the hawk moth, Macroglossum hirundo vitiensis. This was followedby ants and white fly and leaf hopper. Weaver ants were more prevalent in all thedistricts which confirm its host specificity and diversity richness and relative speciesabundance. This was in confirmation with Tan (2001) who reported that Noni hasa symbiotic association in the native range with weaver ants, Oecophylla smaragdina,which provides the ants with food and leaves for nesting in exchange for protectionfrom insect predators.

The Scale insects (Coccids) were found associated with M. citrifolia in moderatedistribution in Kasargod, Kannur and Calicut where as the (Membracids) leafhopper were found in all the sampling sites even though the species abundance wasless in all locations. Grace (2007) reported the incidence of the coccids andmembracids in M. citrifolia as insect pests. The insects identified were given in (Table2).


Intl. J. Noni Res. 2010, 5 (1-2) 53


Herbivore insect abundance and diversity on M. citrifolia compared with naturalenemies in balanced level was observed throughout the sampling sites naturally (Fig.1) which could account for its lower pest abundance in natural vegetation ofMorinda into coastal landscapes (Table 1). Nevertheless, some lepidopteran andhemipteran herbivores clearly use for food and habitat. Insects within the orderHymenoptera (ants and wasps) were also found in greater densities. Furtherinvestigation into herbivore feeding rates, host plant choice and the impact ofherbivores on the performance of M.citrifolia is required for a greater understanding.(Fig. 2)

Some parts of the plant community may not have been affected by the invasion ofinsect pest. There were less significant differences in the insect species assemblagesbetween sampling sites. Diverse array of insects that has colonized these naturalhabitats are not host specific and it demands a quantitative assay to infer their impact.Alternatively, some parts of the native insect community may be generalists and occurin a variety of habitats. Some herbivores may have expanded their host range andincorporated M. citrifolia into their diets. There are numerous records of nativeherbivores switching to neighboring plant species (Hierro et al. 2005) and alsosome documented records of host switching by specialist insects to congeners of thenative plant species (Tallamy 2004).

M. citrifolia may also be an unsuitable host for oviposition, mating and feeding forlarval stages of insect community. Many larval stages of predatory insects requirefood from the host plant or surrounding detritus (McAlpine and Wood 1981).Relatively fewer insect herbivores were found at some sites like Ernakulam andCalicut. Lower herbivore densities were observed on M. citrifolia compared withneighboring plant community. Smaller herbivore densities may be due to plantcompounds and predator richness and abundance of predators feeding onherbivores on M. citrifolia.

This is the first attempt to summarize some of the insect taxonomic and speciesabundance information on M. citrifolia from this coastal line of India (Table 3).This study further stresses the importance of comparing natural plant habitats acrosswest coast of Kerala and insect diversity. The information generated has also allowedexamination of diversity patterns at larger geographical scales. Future studies intothe impacts of insect incidence should compare community composition and trophicdifferences, particularly differences in density and diversity are not detected, or areambiguous.

Acknowledgement

The authors acknowledge the villagers of the surveyed locations for sharing theknowledge on Noni as well as who helped to spot the plants. We thank WNRF for

54 Intl. J. Noni Res. 2010, 5 (1-2)

S.P. Shanthakumar et al., Insect diversity on Morinda citrifolia L. in west coast of Kerala

the financial support and Mr. K. Jayakumar, GIS Lab, MSSRF for his timely help insharing technical information on state topography and sampling sites. We expressour indebtedness to Insect Taxonomist, Dr. V. V. Ramamurthy, Professor and Head,Entomology Division, Indian Agricultural Research Institute (IARI), New Delhi, Dr.Sushila Joshi, In charge of Insect Identification, IARI, Dr. Mahalingam, Head,Entomology Division, Gill Research Institute, Chennai, Dr. B. Vasantharaj David,Chairman, Sun Agro Biotech Pvt. Ltd., Chennai, Dr. S. Sithanantham, Advisor, WNRF,Dr. Subaharan, Head of Entomology Division, and Dr. Dhanapal, Head of AgronomyDivision, Central Plantation Crops Research Institute (CPCRI), Kasaragod, Kerala fortheir support in insect surveillance, identification and intellectual guidance.

Reference

Abbott I. A. La Hawaii traditional Hawaiian uses of plants. V3. Honolulu, Hawaii:Bishop Museum Press; P 97-100.

Grace T. Lim. 2007. Enhancing the weaver ant, Oecophylla smaragdina(Hymenoptera: Formicidae), for biological control of a shoot borer, Hypsipylarobusta (Lepidoptera: Pyralidae), in Malaysian mahogany plantations. Dissertation,Blacksburg, VA.

Hierro J. L., Maron J. L. and Callaway R. M. 2005. A biogeographical approach toplant invasions: the importance of studying exotics in their introduced and nativerange. J. Ecol. 93, 5-15.

Krauss B 1993. Plants in Hawaiian culture. Honolulu: University of Hawaii Press; p103, p 252.

McAlpine J. F. and Wood D. M. 1981. Manual of Nearctic Diptera, Vol. I. ResearchBranch, Agriculture Canada, Ottawa.

Tabrah F L and Eleveth BM 1966. Evaluation of the effectiveness of ancient Hawaiianmedicine. Hawaii Med J. 25: 223-230.

Tallamy D. W. 2004. Do alien plants reduce insect biomassi Conserv. Biol. 18, 1689-1692.

Tan R 2001. Mangrove and wetland wild life at Sungei Buloh Nature Park: GreatMorinda. http://www.naturia.per.sg/buloh/plants/morinda.htm. 3p.

Thomas, C.D., Bulman, C.R. and Wilson, R.J. 2008. Where within a geographic rangedo species survive best ? A matter of scale. Insect Conservation and Diversity, 1, 2-8.

Whistler W 1992. Tongan herbal medicine. Isle Botanica, Honolulu, Hawaii, p. 89-90.

Stampsfiji.com.2002. The Sphingid (hawk) moths of Fiji stamp issue. http://www.stampsfiji.com/stamps/moths/ 4p.

Intl. J. Noni Res. 2010, 5 (1-2) 55

INSTRUCTIONS FOR AUTHORSAIMS AND SCOPEInternational Journal of Noni Research(IJNR) publishes original research andreview articles on all aspects on Noni(Morinda citrifolia L.) and other speciesof Morinda. All submissions will bereviewed by the editorial board or by externalreferences. The journal covers: diversity,cultivation, phytochemistry and clinicalresearch, etc. related to Noni.Three categories of paper will be consideredfor publication in IJNR.1) Reviews2) Full-length papers3) Short communications.SUBMISSION OF MANUSCRIPT (HARDAND SOFT COPY)Authors are advised to submit theirmanuscripts along with a covering letter tothe Editor, International Journal ofNoni Research, # 12, Rajiv Gandhi Road,Sreenivasa Nagar, Chennai - 600 096, IndiaE-MAIL SUBMISSIONAuthors are encouraged to submit theirmanuscripts via e-mail as attachment file tothe E-mail ID : [email protected],[email protected] [email protected] (A coverletter to be sent with the manuscript).PREPARATION OF MANUSCRIPTTwo typed copies of manuscripts using MSWord should be submitted. They should betyped on one side of the paper only, double-spaced with 1.2” margins in all the sides.All pages should be numbered.All the accepted articles will be subjectedto editorial revision. A singed statement fromall the authors should be accompanied withthe manuscript saying that1. the content of the article has not been

published in whole or in part elsewhere2. the article is not currently being

considered for publication elsewhere3. all necessary ethical safeguards have

been met regarding patient and animalexperimentation, etc.

FULL-LENGTH PAPERSThere is no page restriction on overall lengthof article. It should be divided into1) Abstract (100- 200 words) followed by

up to six keywords2) Introduction3) Materials and Methods4) Results5) Discussion6) Acknowledgements7) ReferencesResults and Discussion may be combined.Title page should be prepared in a separate

sheet, which should provide the title ofpaper, name(s) of the author(s), name(s)and address of the institution(s) where thework has been carried out and details ofthe corresponding author with telephoneand fax numbers and e-mail ID.SHORT COMMUNICATIONSIt should be up to 10 double-spacedmanuscript pages with a short summary of50 words. Short communications shouldnot be divided in to different headings.However, headings can be used forAcknowledgements and References. Figuresand Tables should be restricted to amaximum of 2 each. All other style shouldbe as for Full-length papers.FIGURES AND TABLESFigures, figure legends and tables should betyped on a separate sheet and numberedconsecutively in Arabic numerals.Photographs should be sharp with glossyprints. Micrographs, etc. should include abar marker to provide an internal measure ofscale or magnification should be mentionedin each microscopic photograph.REFERENCESReferences in the text should be cited asfollowsSingle author: Surendiran (2004) or(Surendiran, 2004)Two authors: Surendiran and Mathivanan(2005) or (Surendiran and Mathivanan,2005)Three or more authors: Mathivananet al. (2006) or (Mathivanan et al., 2006)Where two or more than two references arequoted consecutively in the text,chronological order should be followed. Ifthe references are within a year, alphabeticalmust be followed. Where references aremade to papers by the same author(s) inthe same year, it should be followed by a, b,c, etc.References must be listed alphabetically bythe name of the authors at the end of themanuscript. The following style must befollowed to cite the referencesJournal articlesAbbott, I.A. 1985. The geographic originof the plants most commonly used formedicine by Hawaiians. Journal ofEthnopharmacology, 14: 213–22.Surendiran, G. and Mathivanan, N. 2006.Antifungal activity of Morinda citrifolia andMorinda pubescens. International Journalof Noni Research, 2: 18-23.Mathivanan, N., Surendiran, G., Srinivasan,K., Sagadevan, E. and Malarvizhi, K. 2005.Review on the current scenario of Noni

research: Taxonomy, distribution, chemistry,and medicinal and therapeutic values ofMorinda citrifolia L. International Journalof Noni Research, 1:1 – 9.Books or monographsLalithakumari, D. 2000. Fungal Protoplast:A Biotechnological Tool. Oxford & IBHPublishing Co., Pvt., Ltd., New Delhi. p184.Single author volumesMathivanan, N. 2004. Current scenario ofthe biocontrol potential of Trichodermafor the management of plant diseases. In:Emerging Trends in Mycology, PlantPathology and Microbial Biotechnology(Eds. Bagyanarayana, G., Bhadraiah, B. andKunwar, I.K.), BS Publications, Hyderabad,India. pp. 364-382.Multi-authors volumesMathivanan, N., Bharati N. Bhat, Prabavathy,V.R., Srinivasan, K. and Chelliah, S. 2003.Trichiderma viride: Lab to land for themanagement of root diseases in differentcrops. In: Innovative Methods andTechniques for Integrated Pest andDisease Management (Eds). Mathivanan,N., Prabavathy, V.R. and Gomathinayagam,S.), Centre for Advanced Studies in Botany,University of Madras, Chennai, India. pp.52-58.Thesis / DissertationSurendiran, G. 2004. Antimicrobialand Wound Healing Activity ofMorinda tinctoria. M. Sc. Thesis,University of Madras, Chennai, India.PROOFSProof will be sent to the correspondingauthor for checking and making essentialcorrections. At this stage, no extensivegeneral revision or alteration will beallowed. Proof should be corrected andreturned to the editor within 5 days ofreceipt.OFFPRINTSCorresponding author will receive the PDFformat of the article by e-mail free of charge.A copy of the journal will be sent by post tothe corresponding author after publication.PAGE CHARGESNo page charge for publishing the paper inthe International Journal of Noni Research.COPYRIGHTWorld Noni Research Foundation is holdingthe copyright of all the papers that arepublished in the International Journal ofNoni Research. Authors may use the materialelsewhere after publication and authors arethemselves responsible for obtainingpermission to reproduce copyright materialfrom other sources.

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