Phytochemical Constituents and Activities of Morinda citrifolia L

Both artificial and naturally occurring antioxidants have been reported to play major roles in protecting membranes and tissues from free radical and xenobiotic-induced oxidative damage (Burton, 1989; Carini et al., 1990). Most living organisms harbour both enzymatic and non-enzymatic systems that protect them against excessive reactive oxygen species. However, various external factors (smoke, diet, alcohol and some drugs) and aging decrease the efficiency of these protective systems, thereby disrupting the redox equilibrium that is established under healthy conditions. Thus, antioxidants that scavenge reactive oxygen species may be of great value in preventing the onset and propagation of oxidative diseases (Willet, 1994). Recently, more attention has been paid to the role of natural antioxidants, mainly phenolic compounds, which may have higher antioxidant activity than do conventional antioxidants, such as vitamins C, E and β-carotene (Vinson et al., 1995). The anti-oxidative effects of natural phenolic compounds, in pure form or in extracts from various plant sources (vegetables, fruits and medicinal plants), have been studied in vitro using a variety of model systems (Meyer et al., 1998; Pietta et al., 1998; Yen & Hsieh, 1998). Therefore, antioxidants, which can neutralize free radicals, may be of central importance in the prevention of carcinogenicity, cardiovascular disease and neurodegenerative changes associated with aging (Halliwell, 1994; Yu, 1994). Epidemiological studies have shown that the consumption of vegetables and fruits can protect humans against oxidative damage by inhibiting and/or quenching free radicals and reactive oxygen species (Ames et al., 1993).


Introduction
Both artificial and naturally occurring antioxidants have been reported to play major roles in protecting membranes and tissues from free radical and xenobiotic-induced oxidative damage (Burton, 1989;Carini et al., 1990).Most living organisms harbour both enzymatic and non-enzymatic systems that protect them against excessive reactive oxygen species.However, various external factors (smoke, diet, alcohol and some drugs) and aging decrease the efficiency of these protective systems, thereby disrupting the redox equilibrium that is established under healthy conditions.Thus, antioxidants that scavenge reactive oxygen species may be of great value in preventing the onset and propagation of oxidative diseases (Willet, 1994).Recently, more attention has been paid to the role of natural antioxidants, mainly phenolic compounds, which may have higher antioxidant activity than do conventional antioxidants, such as vitamins C, E and -carotene (Vinson et al., 1995).The anti-oxidative effects of natural phenolic compounds, in pure form or in extracts from various plant sources (vegetables, fruits and medicinal plants), have been studied in vitro using a variety of model systems (Meyer et al., 1998;Pietta et al., 1998;Yen & Hsieh, 1998).Therefore, antioxidants, which can neutralize free radicals, may be of central importance in the prevention of carcinogenicity, cardiovascular disease and neurodegenerative changes associated with aging (Halliwell, 1994;Yu, 1994).Epidemiological studies have shown that the consumption of vegetables and fruits can protect humans against oxidative damage by inhibiting and/or quenching free radicals and reactive oxygen species (Ames et al., 1993).
Oxidative stress occurs commonly in living organisms, and it is involved in the pathology of cancer, arteriosclerosis, malaria, and rheumatoid arthritis.Moreover, it may play a role in neurodegenerative diseases and ageing processes.It has been demonstrated that many vegetables, fruits, medicinal plants and other foods contain compounds with bioactivity against oxidative stress, and this activity has been attributed to vitamin C, vitamin E, αtocopherol, -carotene, and polyphenolic compounds (Krishnaiah et al., 2011;Moure et al., 2001).Therefore, research regarding natural antioxidants from foods and plants, particularly from folk medicinal plants, is receiving increasing attention throughout the world.

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Nevertheless, extraction of anthraquinones directly from plant roots is still more widely conducted and is conventionally performed by solvent extraction.Other techniques, which include super critical carbon dioxide extraction, subcritical water extraction, ultrasonicassisted extraction (UAE), and microwave-assisted extraction (MAE) have also become of interest as alternatives to the conventional methods.

Plant description
The genus Morinda (Rubiaceae), which includes the species M. Citrifolia L., is made up of around 80 species.M. Citrifolia is a bush or small tree, 3-10 m tall, with abundant broad elliptical leaves (5-17 cm length, 10-40 cm width).

Fig. 1. Unripe fruit
The small tubular white flowers are grouped together and inserted on the peduncle.The petioles leave ring-like marks on the stalks and the corolla is greenish-white (Morton, 1992;Elkins, 1998;Dixon et al., 1999;Ross, 2001;Cardon, 2003).The M. citrifolia L. fruit (3-10 cm length, 3-6 cm width) is ovular and fleshy with an embossed appearance (Fig. 1).It is slightly wrinkly, semi-translucent, and ranges in colour from green to yellow to almost white at the time of picking.It is covered with small reddish-brown buds containing the seeds.The ripe fruit emits a strong butyric acid-like, rancid smell (Morton, 1992;Dixon et al., 1999).The pulp is juicy and bitter, a light dull yellow or whitish colour, and gelatinous when the fruit is ripe; numerous hard triangular reddish-brown pits are found, each containing four seeds (approximately 3.5 mm) (Dittmar, 1993).Moreover, M. citrifolia L. leaves are well known for their strong antioxidant activity, and they have been shown to be safe in acute, subacute, and subchronic oral toxicity tests on mice (West et al., 2007).Inspired by ancient Polynesian legends, M. citrifolia L. leaves have been developed into therapeutic teas.The leaves are also the source for a variety of other health-promoting commercial products.Commercial M. citrifolia L. leaf products have been available in Japan and United States for more than seven years, used mainly for making infusions.However, some manufacturers produce capsules containing powdered M.

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Phytochemicals -A Global Perspective of Their Role in Nutrition and Health 130 citrifolia L. leaves.The major world-wide source of M. citrifolia L. leaves is French Polynesia because leaves from this nation having undergone a safety evaluation (West et al., 2007).Other sources include Panama, Fiji and Hawaii.
Recently, M. citrifolia L. fruit juice is in high demand as an alternative medicine due to its potential anti-microbial, anti-cancer, anti-inflammatory, and antioxidant effects (Wang et al., 2002).However, scientific evidence for the benefits of M. citrifolia L. fruit juice is still limited.In the past decade, M. citrifolia L. fruit juice has emerged on the worldwide market as a safe and popular health product due to its phytochemicals and nutrients.Written documentation of the consumption of this fruit as a food source precedes the twentieth century.Captain James Cook of the British Navy noted in the late 1700s that the fruit was eaten in Tahiti.An 1866 publication in London explained that M. citrifolia L. fruit was consumed as a food in the Fiji islands.
Later publications describe the use of this fruit as a food throughout the Pacific islands, Southeast Asia, Australia, and India.In Roratonga, "the fruit was often eaten by the natives".Australian Aborigines were reported to be "very fond" of the fruit.In Samoa, M. citrifolia L. fruit was common fare, and in Burma, the fruit was cooked in curries or eaten raw with salt.In 1943, Merrill described M. citrifolia L. as an edible plant in a technical manual of edible and poisonous plants of the Pacific islands, in which the leaves and fruits could be used as emergency food.Abbott also reported that M. citrifolia L. had been used as a food, drink, medicine, and colourful dye.The medicinal history and accumulated scientific studies have revealed and confirmed the Polynesians' claims regarding the health benefits of M. citrifolia L.
M. citrifolia L. has identifiable leaves, white tubular flowers, and a distinctive, ovoid, "grenade-like" yellow fruit.The fruit can grow in size up to 12 cm or more and has a lumpy surface covered by polygonal-shaped sections.The seeds, which are triangular and reddish brown, have an air sac contained at one end, making the seeds buoyant.This could explain, in part, the wide distribution of the plant throughout the Polynesian islands.The mature M. citrifolia L .fruit has a foul taste and odour.M. citrifolia L. is not considered to be at risk in the wild.
The fruit juice of M. citrifolia L. is in high demand in alternative medicine for various illnesses, such as arthritis, diabetes, high blood pressure, muscle aches and pains, menstrual difficulties, headaches, heart disease, Acquired Immune Deficiency Syndrome (AIDS), cancer, gastric ulcers, sprains, mental depression, senility, poor digestion, atherosclerosis, blood vessel problems and drug addiction (Kamiya et al., 2004;Wang et al., 2002).Therefore, one of the challenges in recent years has been to process fruit juice so as to make a more modern drug from a traditional product (Chunhieng et al., 2003).A number of in vitro biological activities have been reported, such as angiogenesis inhibition, antioxidant activity, inhibition of cycloxygenases-1 and -2, and tyrosine kinase inhibition.However, most of these have only been tested with crude extracts or fractions of M. citrifolia L., and the compound(s) responsible for these biological activities have not been fully determined, except for two compounds, neolignan and americanin A, which were identified in an nbutanol-soluble partition of the methanol extract of M. citrifolia L. fruits (Su et al., 2005;Zin et al., 2002).

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Phytochemical Constituents and Activities of Morinda citrifolia L.

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M. citrifolia L. has recently been the object of many claims concerning its nutraceutical properties.Various publications have shown that M. citrifolia L. can be used to relieve multiple diseases, and its registered uses span the Pacific, Asia, and Africa.Two clinical studies have reported that relief from arthritis and diabetes are associated with M. citrifolia L. consumption (Elkins, 1998;Solomon, 1999).These beneficial effects may derive from certain compounds, such as scopoletin, nitric oxide, alkaloids and/or sterols, and they also may be due to the antioxidant potential of M. citrifolia L. As a result of this reputation, consumption of this fruit is currently high not only in the producing countries but also in the United States, Japan and Europe.
In response to this demand, some countries (such as Costa Rica and Cambodia) have increased their cultivation of M. citrifolia L. In these countries, the fruit is often commercialised fresh or as juice in both formal and informal markets, but it is also found as pasteurised juice, either in pure form or in combination with other juices (usually grape or blackberry juice).Commercial interest in M. citrifolia L. has increased tremendously in recent years, as indicated by the number of patents registered.In the United States alone, 19 patents have been registered by the US patent and Trademark Office since 1976 (USPTO, 2005).M. citrifolia L. juice has been accepted by the European Union as a novel food (European Commission, Scientific Committee for Food, 2002).Nevertheless, despite the real market opportunities, there has been little scientific research addressing the actual nutritional and functional properties of M. citrifolia L. products.
Several classes of compounds have been isolated from M. citrifolia L., including amino acids, anthraquinones, coumarins, fatty acids, flavonoids, iridoids, lignans and polysaccharides (Chan-Blan-co et al., 2006).Among these, scopoletin, a coumarin derivative, is one of the representative ingredients in M. citrifolia L. Its contribution to anti-microbial, antiinflammatory, and antioxidative activities has been well elucidated (Deng et al., 2007).Samoylenko et al. (2006) recommended scopoletin as a constituent marker for M. citrifolia L. quality control.This compound has been ubiquitously found in M. citrifolia L. collected from Atlantic and Pacific regions and in all examined squeezed fruit juices.Determination of scopoletin in M. citrifolia L. might help to control the quality of M. citrifolia L. products.However, no reports have evaluated the antioxidative activity of scopoletin and other coumarin derivatives in M. citrifolia L.
Many reports on the antioxidative activity of M. citrifolia L. itself have been published.Assays of free-radical-scavenging activity with 1,1-diphenyl-2-picrylhydrazyl (Su et al., 2005;Yang et al., 2007), inhibition of copper-induced low-density lipoprotein oxidation (Kamiya et al., 2004), nitric oxide scavenging activity (Basu & Hazra, 2006) and quenching of H 2 O 2 (Chong et al., 2004;Jeffers et al., 2007) have been performed to evaluate the antioxidative effects of M. citrifolia L. and its products.Polyphenols, reducing glycosides (Calzuola et al., 2006), lignin derivatives (Su et al., 2005) and anthraquinones (Chong et al., 2004) have been suggested as sources of antioxidative activity in M. citrifolia L. However, there is little available information with which to quantitatively evaluate the antioxidative activity of M. citrifolia L ingredients.Recently, a correlation between total phenol and freeradical-scavenging activity was reported (r=0.41,Yang et al., 2007).
The chemical components of M. citrifolia L. have not been well studied, and several anthraquinones and asperuloside are all that have been previously isolated (Levand & Larson, 1979;Srivastava & Singh, 1993).For centuries, scientists and medical professionals have been investigating the chemical constituents in all parts of M. citrifolia (Noni or Yor), including leaves, fruit, bark and roots.The plants contain several medicinally active components that exhibit various therapeutic effects.These include anti-bacterial, anti-viral and anti-cancer activities as well as analgesic effects.Critical reviews of the therapeutic properties of the plants are given by Chan-Blanco et al. (2006) and Wang et al. (2002).Anthraquinones have been shown to be responsible for the therapeutic properties of M. citrifolia L., and among this group of compounds, damnacanthal, which is present mainly in the roots, is of particular interest due to its important anticancer activity (Hiramatsu et al., 1993).
Flavonol glycosides appear to predominate in M. citrifolia L. leaves; rutin and other flavonol glycosides have previously been identified in raw M. citrifolia L. leaves (Sang et al., 2001a).However, the presence of flavonol aglycones in M. citrifolia L. leaves has not been previously reported.Flavonoids have been indicated to possess a variety of biological activities (Garcia-Mediavilla et al., 2006;Kampkotter et al., 2007), and they may play an important role in M. citrifolia L. leaves.To date, there has been no validated analytical method for determining the flavonol constituents of M. citrifolia L. leaves.

Yield
M. citrifolia L. is a perennial bush, and it is possible to find fruits at different stages of maturity on the same plant at the same time.The species is generally found from sea level to 400 m, although it adapts better to coastal regions (Luberck & Hannes, 2001).Under favourable conditions, the plant bears fruit approximately nine months to one year after planting.At this stage, the fruits can be harvested, but they are generally small, and the yield per tree is low.Some producers choose not to harvest in the first year, and they prune in order to let the bush grow stronger.In Hawaii, M. citrifolia L. fruits are harvested throughout the year, although there are seasonal patterns in flowering and fruit bearing (meteorological factors, fumigation, and irrigation) (Nelson, 2001(Nelson, , 2003)).
In Hawaii, M. citrifolia L. plots are usually harvested two or three times per month, although fruit production is lower during winter.With a density of 638 plants per hectare; good soil fertility, drainage, and irrigation; appropriate pest, disease and weed control; and an appropriate fertilisation plan, it is possible to obtain yields of 7 tonnes/ha/year after the fifth year (Nelson, 2001(Nelson, , 2003)).With a juice extraction rate of approximately 50% (w/w), one hectare can thus yield around 35 tons of juice.However, many factors may affect these yields, and most producers do not obtain such good results because of diseases and/or poor agricultural practices (growing wide plants).In Hawaii, an average annual yield of 50 tonnes/ha is generally attained (Nelson, 2001(Nelson, , 2003)).Depending on the post-harvest technology programme adopted, the fruits may be harvested at different stages of development and continue to mature.The evolution of the colour and firmness of fruits left to ripen naturally on the tree is reported in Table 1.Nonetheless, most processors buy M. citrifolia L. harvested at the "hard white" stage for juice production as the fruits become soft too quickly once this stage is reached (Nelson, 2001(Nelson, , 2003)).The change from stage 4 to 5 occurs very quickly (within a few hours), and the pulp practically liquefies and turns from green to white, as well as develops the characteristic butyric smell.

Maturity stage
The fruits are individually selected on the tree and harvested by hand.At the "hard white" stage, they are well able to withstand being transported in baskets or containers, and exposure of the fruits to light or high temperatures immediately after harvest does not affect their overall quality.Before processing, fruits are ripened at room temperature for one day or more, depending on the end product (such as tea, juice, pulp, dietetic products) (Nelson, 2003).

Chemical composition of M. citrifolia L.
Approximately 160 phytochemical compounds have already been identified in the M. citrifolia L. plant, and the major micronutrients are phenolic compounds, organic acids and alkaloids (Wang & Su, 2001).Of the reported phenolic compounds, the most important are anthraquinones (such as damnacanthal, morindone, morindin.)and also aucubin, asperuloside, and scopoletin (Wang & Su, 2001).The main organic acids are caproic and caprylic acids (Dittmar, 1993), whereas the principal reported alkaloid is xeronine (Heinicke, 1985).Chan-Blanco et al. (2006) reviewed the chemical constituents of different parts of the plant (Table 2).
However, chemical composition differs significantly according to the part of the plant.The complete physico-chemical composition of the fruit has not yet been reported, and only partial information is available on M. citrifolia L. juice.The fruit contains 90% water, and the main components of the dry matter appear to be soluble solids, dietary fibre and proteins (Chunhieng, 2003).The fruit's protein content is surprisingly high, representing 11.3% of the juice dry matter, and the main amino acids are aspartic acid, glutamic acid and isoleucine (Chunhieng, 2003).
Minerals account for 8.4% of the dry matter, and these minerals are mainly potassium, sulphur, calcium and phosphorus; traces of selenium have been reported in the juice (Chunhieng, 2003, Table 3).Vitamins have been reported in the fruit, mainly ascorbic acid (24-158 mg/100 g dry matter) (Morton, 1992;Shovie & Whistler, 2001) and provitamin A (Dixon et al., 1999).Phenolic compounds have been found to be the major group of functional micronutrients in M. citrifolia L. juice: damnacanthal, scopoletin, morindone, alizarin, aucubin, nordamnacanthal, rubiadin, rubiadin-1-methyl ether and other anthraquinone glycosides have been identified in M. citrifolia L. (Morton, 1992;Dittmar, 1993;Dixon et al., 1999;Wang & Su, 2001).Damnacanthal is an anthraquinone that has been characterised and has some important functional properties (mainly anti-carcinogenic) (Solomon, 1999).Scopoletin is a coumarin that was isolated in 1993 at the University of Hawaii and has been found to have analgesic properties as well as a significant ability to control serotonin levels in the body (Levand & Larson, 1979).Other researchers have shown that scopoletin may also have anti-microbial (Duncan et al., 1998) and anti-hypertensive effects (Solomon, 1999).
Multiple Hawaiian teams (Heinicke, 1985;Solomon, 1999) have reported the presence of a novel component, proxeronine, which is an alkaloid that is claimed to combine with human proteins and improve their functionality.The above authors attribute most of the beneficial effects of M. citrifolia L. to xeronine.Nonetheless, neither the chemical characterisation of this alkaloid nor the method used to assess its levels has been published to date.
A research group led by Chi-Tang Ho at Rutgers University in the United States (US) is searching for new novel compounds in the M. citrifolia L. plant.They have successfully identified several new flavonol glycosides, an iridoid glycoside from M. citrifolia L. leaves, a trisaccharide fatty acid ester, rutin, and an asperulosidic acid from the fruit.Two novel glycosides and a new unusual iridoid named citrifolinoside have been shown to have an inhibitory effect on AP-1 transactivation and cell transformation in the mouse epidermal JB6 cell line.James Duke listed 23 different phytochemicals found in M. citrifolia L. as well as 5 vitamins and 3 minerals in an authoritative handbook of phytochemicals.

Xeronine system
Retired biochemist Ralph Heinicke states that M. citrifolia L. fruit contains a natural precursor of xeronine that he named Proxeronine.Proxeronine is converted to the alkaloid xeronine in the body by an enzyme he named proxeroninase.His hypothesis is that xeronine is able to modify the molecular structure of proteins.Thus, xeronine has a wide range of biological activities.When a protein such as an enzyme, receptor, or signal transducer is not in the appropriate conformation, it will not work properly.Xeronine will interact with the protein and make it fold into its proper conformation.The result is a properly functioning protein.Whenever a problem arises in the cell due to a structural problem with a protein, xeronine's presence would be beneficial.His hypothesis may explain why Tahitian Noni @ juice (TNJ) can help in many health problems in different ways.He has obtained several patents for xeronine.He states that the active ingredient in many of the effective folklore drugs is xeronine.This alkaloid is a critical normal metabolic coregulator.The ailments that he believes are helped by M. citrifolia L. include high blood pressure, menstrual cramps, arthritis, gastric ulcers, sprains, injuries, mental depression, senility, poor digestion, drug addiction, and pain."I have devoted much of my life to the study of this unique substance that I have named 'xeronine'.I am convinced of the tremendous benefits achieved by furnishing the body with a proper supply of this material" (Heinicke, 2001).

2.4
Biological activity of M. Citrifolia L.

Anti-microbial effects
The anti-microbial activity of M. citrifolia L. may have been its first observed property; indeed, the fruit contains relatively large amounts of sugars that do not ferment even when fruits are stored in closed containers at ambient temperature.This property is used to www.intechopen.comPhytochemical Constituents and Activities of Morinda citrifolia L.

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transport the fruit by boat from the scattered Pacific islands to processing plants without specific treatment.
It has been reported that M. citrifolia L. inhibits the growth of certain bacteria, such as staphylococcus aureus, Pseudomonas aeruginosa, Proteus morgaii, Bacillus subtilis, Escherichia coli, Helicobacter pylori, Salmonella and Shigella (Atkinson, 1956).The same author claims that the anti-microbial effect observed may be due to the presence of phenolic compounds such as acubin, l-asperuloside, alizarin, scopoletin and other anthraquinones.
Another study showed that an acetonitrile extract of the dried fruit inhibits the growth of Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, and Streptococcus pyrogene (Locher et al., 1995).
Other studies have reported a significant antimicrobial effect on various strains of Salmonella, Shigella, and E. coli (Bushnell et al., 1950;Dittmar, 1993).Furthermore, they showed that the anti-microbial effect is highly dependent on the stage of ripeness and on processing, being greater when the fruit is ripe and undried.

Anti-cancer activity
The immunomodulatory properties (the capacity to enhance the host immune system) of M. citrifolia L. juice have been studied by a Japanese research team (Hirazumi et al., 1996;Hirazumi & Furusawa, 1999).The ethanol precipitable fraction (ppt) of M. citrifolia L. juice, corresponding to a polysaccharide-rich substance composed of glucuronoic acid, galactose, arabinose and rhamnose, has been found to have immunomodulatory and anti-tumour effects against Lewis lung carcinoma (LLC).In cell models, M. citrifolia L.-ppt seems to stimulate the production of T-cells, thymocytes and macrophages that produce cytokines, which are important mediators of tumour cytostasis and cytotoxicity.
M. citrifolia L.-ppt also appears to stimulate murine effector cells to release several mediators such as cytokines.These mediators slow down the cell cycle in tumours, increase the response of cells to other immunised cells that fight tumour growth and have potent macrophage activator activity suspected of playing a role in the death of tumours (Hirazumi et al., 1996;Hirazumi & Furusawa, 1999).
In the same study, mice were inoculated with LLC, and those ingesting a daily dose of 15 mg of M. citrifolia L. juice showed a significant increase (119%) in lifespan.Nine out of 22 mice with terminal cancer survived for more than 50 days.In addition, the ingestion of M. citrifolia L.-ppt combined with conventional chemotherapy proved to increase the lifespan of mice with cancer, (Hirazumi et al., 1994).
Another Japanese team studied the influence of damnacanthal, an anthraquinone extracted from a chloroform extract of M. citrifolia L. fruits.Surprisingly, the researchers found that damnacanthal induces normal morphology in a particular type of cell found in human neoplasias (K-ras-NKR cells) that multiply uncontrollably and are highly malignant (Hiramatsu et al., 1993).

Phytochemicals -A Global Perspective of Their Role in Nutrition and Health 140
Another study showed that commercial M. citrifolia L. juice (Tahitian Noni juice) prevents the formation of chemical carcinogen-DNA-adducts.In the above study, rats with artificially induced cancer in specific organs were fed for one week with 10% M. citrifolia L. juice in their drinking water and rat food (rat chow) ad libitum.They showed reduced DNA-adduct formation depending on sex and organ.The reduction rates were: in female rats, heart 30%, liver 42%, lungs 41% and kidneys 80%; in male rats, heart 60%, liver 70%, lungs 50% and kidneys 90% (Wang & Su, 2001).

Anti-oxidant properties
The antioxidant properties of ethanol and ethyl acetate extracts of M. citrifolia L. fruit have been assessed using the ferric thiocyanate method (FTC) and thiobarbituric acid test (TBA).
The authors found that ethyl acetate extract strong inhibited lipid oxidation, comparably to the same weight of pure α-tocopherol and butylated hydroxyl toluene (BHT) (Mohd et al., 2001).
Radical scavenging activity was also measured in vitro by the tetrazolium nitroblue (TNB) assay in commercial juice by assessing the capacity of the juice to protect cells and lipids from oxidative alteration promoted by superoxide anion radicals (SARs).The SAR scavenging activity of M. citrifolia L. juice was shown to be 2.8 times higher than that of vitamin C, 1.4 times that of pycnogenol (PYC) and almost the same magnitude as that of grape seed powder (Wang & Su, 2001).

Anti-inflammatory activity
The anti-inflammatory activity of an aqueous extract from M. citrifolia L.-juice was observed by inducing a locally acute inflammatory response with the help of a pro-inflammatory agent (bradykinin).It was found that the oral administration of M. citrifolia L. juice extract (200 mg) rapidly inhibits the formation of rat paw oedema.This effect may have resulted from interference with the B2 receptor-mediated mechanism by which bradykinin induces rat paw oedema (Mckoy et al., 2002).
Another study showed that commercial M. citrifolia L. juice selectively inhibits cyclooxygenase enzymes (COX-1 and COX-2) involved in breast, colon and lung cancer and also has anti-inflammatory activity (Su et al., 2001).The ability of noni juice to inhibit these enzymes was compared to that of traditional commercial non-steroidal inflammatory drugs, such as aspirin, Indomethacin and Celebrex.M. citrifolia L. juice showed selective inhibition of COX activity in vitro and a strong anti-inflammatory effect comparable to that of Celebrex, and presumably, this juice lacks side effects.

Analgesic activity
Recent research has examined the analgesic properties of commercial juice in rats.The results showed that rats fed 10% or 20% M. citrifolia L. juice had greater pain tolerance (162% or 212%, respectively) compared with the placebo group (Wang et al., 2002).A French research team has also studied the analgesic and sedative effects of M. citrifolia L. on mice through the writhing and hotplate tests.M. citrifolia L. root extract (1600 mg/kg) showed significant analgesic activity in the animals, similar to the effect of morphine (75% and 81% protection using M. citrifolia L. extract and morphine, respectively), and it also proved to be non-toxic (Younos et al., 1990).

Cardiovascular activity
Recent research has demonstrated the ability of M. citrifolia L. fruit to prevent arteriosclerosis, a disease related to the oxidation of low density lipoproteins (LDLs).
Methanol and ethyl acetate extracts showed 88% and 96% inhibition, respectively, of copperinduced LDL oxidation by the thiobarbituric acid relative substance method.This beneficial effect could be due to the presence of lignans, which are phenylpropanoid dimers (Kamiya et al., 2004).

Antibacterial activity
Acubin, l-asperuloside, alizarin in M. citrifolia L. fruit, and certain other anthraquinone compounds in M. citrifolia L. roots, are all proven antibacterial agents.These compounds have been shown to fight infectious bacteria such as Pseudomonas aeruginosa, Proteus morgaii, Staphylococcus aureus, Bacillis subtilis, Escherichia coli, Salmonella, and Shigela.These antibacterial compounds in M. citrifolia L. are responsible for the treatment of skin infections, colds, fevers, and other bacteria-caused health problems.Bushnell reported on the antibacterial properties of certain plants found in Hawaii, including M. citrifolia L.He further reported that M. citrifolia L. was traditionally used to treat broken bones, deep cuts, bruises, sores and wounds.Extracts from the ripe M. citrifolia L. fruit exhibit moderate antibacterial properties against Salmonella typhosa.Salmonella montevideo, Salmonella schottmuelleri, Shigella paradys, BH and Shigella paradys, III-Z.Leach demonstrated that acetone extracts obtained from Cycas circinalis, M. citrifolia, Bridelia penangiana, Tridax Procumbens, Hibiscus tiliaceus, and Hypericum papuanun show antibacterial activity.The widespread medicinal use of these plants would suggest that they do contain pharmacologically active substances, and alternative methods of extraction and screening should be utilised to find the major bioactive component in the plants for the purpose of new drug development.Locher reported that selected plants including M. citrifolia have a history of use in Polynesian traditional medicine for the treatment of infectious diseases.These plants have been investigated for anti-viral, anti-fungal, and anti-bacterial activity in vitro.
Their study using in vitro biological assays confirmed that some of the Hawaiian medicinal plants in ethnobotanical reports have curative properties against infectious diseases.
Duncan demonstrated that scopoletin, a health promoter in M. citrifolia L., inhibits the activity of E. coli, which is associated with recent outbreaks resulting in hundreds of serious infections, even death.M. citrifolia L. also helps stomach ulcers by inhibiting H. pylori bacteria.

Antiviral activity
Umezawa and coworkers found that a compound isolated from M. citrifolia L. roots named 1-methoxy-2-formyl-3-hydroxyanthraquinone suppresses the cytopathic effect of HIVinfected MT-4 cells without inhibiting cell growth.

Anti-tubercular effects
At the International Chemical Congress of the Pacific Basin Societies meeting in Honolulu, Saludes and colleagues from the Philippines reported that M. citrifolia L. kills Mycobacterium tuberculosis.A concentration of extracts from M. citrifolia L. leaves killed 89% of the bacteria in a test tube, almost as effectively as the leading anti-TB drug Rifampicin, which has an inhibitory rate of 97% at the same concentration.Although there have been anecdotal reports of native use of M. citrifolia L. in Polynesia as a medicine against tuberculosis, this is the first report demonstrating the antimycobacterial potential of compounds obtained from M. citrifolia L. leaves."I hope that pharmaceutical companies will pay attention to this research and explore the M. citrifolia L. plant as a potential source of drugs", said Saludes in Manila.

Antitumor activity
At the 83 rd Annual meeting of the American Association for Cancer Research in 1992, Hirazumi, a researcher at the University of Hawaii, reported that the alcohol-precipitate of M. citrifolia L. fruit juice (noni-ppt) has anticancer activity on lung cancer in C57 B1/6 mice.This M. citrifolia L.-ppt was shown to significantly prolong (by up to 75%) the life of mice with implanted Lewis lung carcinoma.It was concluded that the M. citrifolia L.-ppt seems to suppress tumour growth indirectly by stimulating the immune system.Improved survival time and curative effects occurred when M. citrifolia L.-ppt was combined with sub-optimal doses of the standard chemotherapeutic agents, such as adriamycin (Adria), cisplatin (CDDP), 5-fluorouracil (5-FU) and vincristine (VCR), suggesting that M. citrifolia L.-ppt has important clinical utility as a supplemental agent in cancer treatment.These results indicate that M. citrifolia L.-ppt may enhance the therapeutic effect of anticancer drugs.Therefore, it may be of benefit to cancer patients by enabling them to use lower doses of anti-cancer drugs to achieve the same or even better results.
Dr. Wang and coworkers demonstrated a cytotoxic effect of TNJ on a cultured leukaemia cell line at various concentrations.TNJ showed dose-dependent cytotoxicity on cultured cancer cells by inducing cancer cell necrosis at high doses and apoptosis at lower doses.Synergistic effects of TNJ with known anticancer drugs have been found.At sub-optimal doses, both prednisolone and TNJ can induce apoptosis.When the dose of prednisolone is fixed and the dose of TNJ increases, apoptotic cells significantly increase.Therefore, TNJ is able to enhance the efficacy of anticancer drugs such as prednisolone.When a single dose of Taxol induces a lower percentage of apoptosis in leukaemia cells, TNJ enhances the rate of apoptosis to 100%.These results indicate that TNJ is able to enhance the therapeutic effects of anticancer drugs such as Taxol.These findings regarding the combination of anticancer drugs with TNJ may be significant.This approach may allow lower doses of synthetic anticancer drugs to be used, increase the tolerance of patients to the toxicity of anticancer drugs, and increase immune function, thus creating a new method for treating cancer patients.
In 1993, Hiramatsu and colleagues reported in Cancer Letters the effects of over 500 extracts from tropical plants on K-Ras-NRK cells.Damnacanthal, isolated from M. citrifolia L. roots, is an inhibitor of Ras function.The ras oncogene is believed to be associated with signal transduction in several human cancers such as lung, colon, and pancreatic cancer and leukaemia.
Hiwasa and coworkers demonstrated that damnacanthal, an anthraquinone compound isolated from the M. citrifolia L. roots, has potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptors.In his research, he examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells.Consequently, the ultraviolet light induced a concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases.
After pretreatment with damnacanthal, there was a stimulatory effect on ultraviolet-induced apoptosis.
Dong reported that two glycosides extracted from M. citrifolia L.-ppt were effective at inhibiting cell transformation induced by TPA or EGF in the mouse epidermal JB6 cell line.This inhibition was found to be associated with the inhibitory effects of these compounds on AP-1 activity.The compounds also blocked the phosphorylation of c-jun, a substrate of JNKs, suggesting that JNKs are a critical target for the compounds in mediating Ap-1 activity and cell transformation.

Antihelmintic activity
An ethanol extract of tender M. citrifolia L. leaves was found to induce paralysis and death in the human parasitic nematode Ascaris Lumbricoides within a day.A botanist via Morton reported that M. citrifolia L. has been used in the Philippines and Hawaii as an effective insecticide.

Hypotensive activity
Dang Van Ho of Vietnam demonstrated that total extract of M. citrifolia L. roots has a hypotensive effect.Moorthy and coworkers found that an ethanol extract of M. citrifolia L. roots lowers blood pressure in anesthetised dogs.Youngken's research team determined that a hot water extract of M. citrifolia L. roots lowers blood pressure in anesthetised dogs.A Hawaiian physician reported that M. citrifolia L. fruit juice has a diuretic effect.

Immunological activity
Asahina found that an alcohol extract of M. citrifolia L. fruit at various concentrations inhibits the production of tumour necrosis factor-alpha (TNF-), which is an endogenous tumour promoter.Therefore, the alcohol extract may inhibit the tumour promoting effect of TNF-.Hirazumi found that M. citrifolia L.-ppt contains a polysaccharide-rich substance that inhibits tumour growth.It does not cause significant cytotoxicity in adopted cultures of lung cancer cells, but it can activate peritoneal exudate cells to impart profound toxicity when co-cultured with tumour cells.This suggests the possibility that M. citrifolia L.-ppt may suppress tumour growth through activation of the host immune system.M. citrifolia L.ppt is also capable of stimulating the release of several mediators from murine effector cells, including TNF-, interleukin-1 beta (IL-1 ), IL-10, IL-12, interferon-gamma (IFN-) and nitric oxide (NO).Hokama separated ripe M. citrifolia L. fruit juice into 50% aqueous alcohol and precipitate fractions, which was found to stimulate BALB/c thymus cells in ( 3 H)thymidine analysis.It has been suggested that inhibition of Lewis lung tumours in mice, in part, may be due to the stimulation of the T-cell immune response.
Wang and coworkers at the University of Illinois college of Medicine observed that the thymus is enlarged in animals treated with TNJ.The wet weight of the thymus was 1.7 times that of control animals on the seventh day after receiving 10% TNJ in drinking water.The thymus is an important immune organ in the body that generates T cells and is involved in the aging process and cellular immune fractions.TNJ may enhance immune function by stimulating thymus growth and thereby exerting anti-aging and anticancer activities and protecting people from other degenerative diseases.

Mental health and improved high frequency hearing
A small human clinical trial of the effects of TNJ on auditory function and quality of life in patients with decreased bone mineral density and auditory function was conducted at the UIC College of Medicine, Rockford, IL.This study showed that TNJ improves both mental health and high frequency hearing.The results suggest that increased amounts or extended duration of TNJ intake may be required to influence this disorder.

Study of TNJ for cancer prevention
"To take medicine only when you are sick is like digging a well only when you are thirstyis it not already too late?"(Chi Po, c 2500 B.C.).This proverb suggests that prevention is more important than treatment.
Cancer is the second leading cause of death in the US.According to the American Cancer society, 1500 people per day die from cancer in the United States.Fighting against cancer is a great task for scientists engaged in this field.The aetiology of most cases of human cancer remains unknown.Exposure to environmental carcinogens accounts for more than 90% of human cancers.Cigarette smoke is the number one high-risk environmental factor.Although some cancers are preventable, a means to prevent most cancers is not yet known.
Seeking a natural way to prevent human cancer is an urgent task for cancer prevention investigators.
Studies of food, diet, and cancer have indicated that lifestyle changes such as eating more fruits and vegetables and quitting smoking will help prevent cancer."A new plate" for America (75% vegetables, 25% meat) appeared at the 2001 annual conference of the American Institute for Cancer Research.Although TNJ possesses a broad range of therapeutic effects, its ability to prevent cancer remains unclear.Recently, new hypothesis has been investigated: whether or not TNJ can help prevent cancer during the early stages of chemical carcinogenesis.
This hypothesis was examined using two carcinogenic animal models and one human clinical study of a group of current smokers.The animal models included the following: the DMBA-induced mammary gland tumourigenesis model and an acute liver injury model induced by the liver carcinogen carbon tetrachloride (CCl 4 ).These are classical extrinsic carcinogenic models.DMBA-induced DNA adduct formation and histological examination by light and electron microscopy were used as sensitive biomarkers to evaluate the preventive effects of TNJ at the initiation stage of multiple-step carcinogenesis.In the mammary breast carcinogenic model, to monitor the mechanisms of carcinogenesis and DMBA DNA-adduct formation in mammary tissue, the focus was on the pathogenic changes after DMBA administration.In the acute liver injury model, the histopathological changes in liver tissue and levels of both super-oxide anion free radicals (SAR) and lipid hydroperoxide (LPO) after CCl 4 administration were the focus.
DMBA DNA-adduct formation was used as a marker to examine whether TNJ is able to prevent carcinogen-induced DNA damage.Most chemical carcinogens need to be activated by endogenous enzymes to be transformed into a form that readily binds to genetic DNA to form DNA-adducts. Carcinogen-DNA adduct formation is an important DNA damage marker that predicts the possibility of cancer development.Most scientists agree that carcinogen-induced DNA adduct formation is an early critical step in the multiple stages of carcinogenesis.Carcinogen-DNA adducts can be repaired by endogenous enzymes.Unrepaired adducts are fixed after one cell cycle.Unrepaired DNA damage is responsible for mutations and subsequent cancer development.Therefore, preventing carcinogen-DNA adduct formation is a key aspect of preventing the initial steps of carcinogenesis.If TNJ can prevent and/or block the formation of carcinogen-induced DNA adducts, it may prevent cancer at the initiation of multiple-stage carcinogenesis.
In recent years, increasing demand for higher quality and safer foods and medicines, as well as concern for environmental pollution during their commercial production, have triggered stringent regulations on toxin levels in foods and medicines as well as on the discharge of pollutants to the environment.In addition, there has been increasing consumer preference for natural substances.All these factors have provided strong motivation for the development of cost-effective new technologies, such as the eco-friendly extraction of natural substances employing green and safe solvents.In recent years, supercritical fluid extraction (SFE) has emerged as a highly promising environmentally benign technology for the production of natural extracts such as flavours, fragrances, spice oils, and oleoresins; natural anti-oxidants; natural colours; nutraceuticals and biologically active compounds.The state of a substance is called supercritical when both temperature and pressure exceed their critical point values.
A supercritical fluid combines two beneficial properties, namely high density (which imparts high solvent power) and high compressibility (which permits high selectivity due to large changes in solvent power in response to small changes in temperature and pressure).
In addition, SFE offers very attractive extraction characteristics owing to its favourable diffusivity, viscosity, surface tension, and other thermo-physical properties.
Since the 1980s, several potential applications of SFE have been reported.So far, the most popular SF has been carbon dioxide (CO 2 ), owing to its easy availability, low cost, nonflammability, nontoxicity, and its possession of a wide spectrum of solvent properties.Its critical temperature is 31.1 °C and its critical pressure is 73.8 bar.Dense or supercritical carbon dioxide could very well be the most commonly used solvent in this century due to its wide-ranging applications.Its near-ambient critical temperature makes it ideally suitable for processing thermally labile natural substances.It is generally regarded as safe (GRAS), and it yields microbial-inactivated, contaminant-free, tailor-made extracts of superior organoleptic profile and longer shelf life with highly potent active ingredients.The SFE technique ensures high consistency and reliability in the quality and safety of bioactive heatsensitive botanical products as it does not alter the delicate balance of bioactivity of natural molecules.All of these advantages are almost impossible with conventional processes.Therefore, SFE technology using SC-CO 2 as the solvent is an ideal alternative to the conventional techniques for the extraction of bioactive ingredients from spices.

Conclusion
Morinda citrifolia L., commonly known as noni, has a long history of widespread use as a food in tropical regions from Indonesia to the Hawaiian Islands, and it is used as an herbal remedy for multiple diseases.Its fruit, leaves, seeds, bark and roots have been traditionally used for the prevention or improvement of various diseases, including arthritis, infections, colds, cancer, and diabetes.It has been found that Morinda citrifolia L., has antioxidant potential equivalent or similar to that of synthetic antioxidants, such as BHT and BHA, which are currently used as food additives.The antioxidants which are found in Morinda citrifolia L., have no side effects, and thus they could replace synthetic antioxidants in the food processing industry and have potential for use in preventive medicine.Thus, this fruit can be used as an antioxidant additive in the food processing industry.