NTRODUCTION:

Reactive oxygen species are unstable and highly reactive substances that cause oxidative damage into the cells. They are thought to be very harmful to human health as they cause great damage to cell membranes through membrane lipid peroxidation and decreased membrane fluidity and also DNA mutations leading to many diseases such as cancer, coronary diseases, arteriosclerosis, inflammatory disorders, diabetics, etc.¹. Antioxidants are compounds that can delay or inhibit the oxidation of biological molecules by inhibiting either the initiation or propagation of oxidative chain reactions or enhancing our cellular antioxidant defenses². The common mechanism of action of antioxidant compounds is mediated through direct interaction with free radicals to stabilize them and hence also known as free radical scavengers.

Antioxidative activity of plant secondary metabolites is based on their ability to donate hydrogen atom to free radicals. Recently more attention has been paid to the role of natural antioxidants present in medicinal plants which may have higher antioxidant activity than conventional vitamins³. The potential of the antioxidant constituents of plant materials for the maintenance of health and protection from coronary heart diseases and cancer is also raising interest among scientists and food manufacturers as consumers move toward functional foods with specific health benefits⁴.

Morinda citrifolia (Rubiaceae), commonly known as Noni or Indian Mulberry, is a tropical and subtropical plant grown in the pacific islands. It is considered to be the secondmost important and popular medicinal plant in Polynesian countries due to its greater number and variety of health giving phytochemicals. It has been reported to have a broad range of therapeutic effects including antibacterial, antifungal, antitumour, antihelminthic, analgesic, hypotensive, anti-inflammatory and immune enhancing effects⁵. The methanol fraction of Morinda citrifolia fruits has been previously shown to be more effective in suppressing the proliferation of cancer cells than other extracts. The activity of this fraction was also selective to cancer cells as no significant toxicity to normal cells was recorded⁶. Due to the wide range of therapeutic benefits reported for Morinda citrifolia, the plant has recently gained a great deal of interest by scientists and medical professionals⁷. Furthermore, Noni plant and its products have been officially designated as G.R.A.S. by the F.D.A. and Department of Agriculture in U.S. Hence, there has been no restriction in the use of Noni products as health tonics and other alternative medicines. Also, the chromatographic fractions obtained from root, fruit and leaf of Morinda citrifolia showed the antioxidant activity⁸.

It has now become very essential to explore natural and more economic antioxidant compounds or secondary metabolites to overcome the toxicity and higher manufacturing cost of synthetic antioxidants. There is also a great need to produce promising natural lead compounds on large scale, independent from environmental factors. Plant cell cultures have been considered as the attractive alternatives for the production of secondary metabolites⁹. Out of all in vitro techniques, suspension cultures are more advantageous to produce important bioactive compounds on large scale in less time period and so they have been widely used since they provide a homogeneous system allowing easy environmental control of the cells. The use of such technique for plants like Morinda citrifolia L. is highly valued as the plant has previously been shown to be a good source of various antioxidants.

Since our literature survey revealed that no antioxidant or antiproliferative activity of in vitro culture of Morinda citrifolia L. had ever been undertaken, the primary objectives of the present study was to test the methanolic extract of suspension culture derived from the root explant using convenient in vitro assays. Given that the antioxidant effects of medicinal plants are attributed mainly due to the presence of flavonoids¹⁰ and phenolics¹¹, we also studied the flavonoid and/or phenolic content of Morinda citrifolia root culture.

MATERIALS AND METHODS:

Plant material and suspension culture:

The plant specimens of Morinda citrifolia were collected from Vidarbha region of Maharashtra, State of India. After the plants were identified and authenticated by the agriculture experts, voucher specimens were deposited in our laboratory collections (Department of Biotechnology, Sant Gadge Baba Amravati University).

Chemical reagents:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), Glutathione, foetal bovine serum (FBS), RPMI-1640, quercetin, gallic acid and antibiotics were purchased from HiMedia Laboratories. Alamar Blue^TM was purchased from AbD Serotec (Kidlington, UK). All the solvents and other chemicals were of the highest analytical grade and were purchased from local sources.

Cell suspension culture:

Cells of Morinda citrifolia were obtained from friable callus derived from root explants and maintained in 250 ml Erlenmeyer flasks containing 100 ml of the MS medium¹² supplemented with 2 mg/l NAA (Naphthalene Acetic Acid), 1 mg/ml BA (Benzyl Adenine), 0.2 mg/ml Kinetin and 20 g/l sucrose. The cell suspension cultures were maintained in orbital shaking incubator (120 rpm) at 25 ± 2ºC under dark conditions. Subculturing of cell cultures was done after every 15 days.

Preparation of extract:

The suspended cells in suspension culture were harvested by filtration using a Buchner funnel with filter paper (Whatman No. 4) and kept in an oven at 60ºC for 2 hrs. The dried powder (20 mg) of root suspension culture cells was taken and suspended in 10 ml of methanol for 24 hrs and then kept in boiling water bath for next 10 min and finally sonicated for 15 minutes. The extract was centrifuged at 3000 rpm for 20 minutes and supernatant was evaporated to dryness. After evaporation, the methanol extract of the sample was collected and dissolved in methanol to make the stock solution of 1mg/ml. The yield of extract was found to be 29 %. Antioxidant assays

DPPH photometric assay:¹³

The sample stock solution (1 mg/ml) was diluted to final concentrations of 500, 250, 100 and 50 µg/ml in methanol. A total of 1 ml of a freshly prepared DPPH solution (20 mg/l) was added to 500 µl of sample solution of different concentrations and allowed to react at room temperature for 30 minutes in dark condition. After 30 min., the absorbance was measured at 517 nm and converted into the percentage antioxidant activity using the following formula:

DPPH radical scavenging activity (%) =

[(A_control – A_extract)/A_control] ×100

Methanol plus plant extract solution was used as a blank while DPPH solution plus methanol was used as a negative control. The positive control was DPPH solution plus glutathione and ß-carotene samples. The IC₅₀ values were calculated by using nonlinear regression analysis of dose dependent curves. IC₅₀ value is the sample concentration required to scavenge 50% of the DPPH free radicals.

Reducing power assay:¹⁴

Different concentrations of methanol extract of root suspension culture (50-500 µg/ml) were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide (2.5 ml, 1%). The mixture was incubated at 50ºC for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and ferric chloride (0.5 ml, 0.1%) and the absorbance was measured at 700 nm and compared with ascorbic acid as a standard. Increased absorbance of the reaction mixture indicated increased reducing power.

Cell culture:

Cytotoxicity of root suspension extract was performed on B16 (mouse melanoma) and MCF-7 (human breast cancer) cell lines. B16 and MCF7 cell lines were grown and maintained in RPMI 1640 medium supplemented with 10 % (v/v) heat inactivated foetal bovine serum (FBS) and penicillin (50 U/mL), Streptomycin (50 μg/mL). Cells were maintained in a humidified atmosphere of 95 % air and 5 % CO₂at 37°C.

Cell viability and cytotoxicity assay:

Antiprolifereative activity of the root suspension culture extract of Morinda citrifolia was quantified by the MTT assay¹⁵ with some modifications or Alamer Blue assay¹⁶. The B16 and MCF-7 cancer cell lines were seeded in 96-well plates at a density of 5,000 cells per well and cultures were allowed to establish by incubating plates at 37ºC for 24 hrs. After cells were treated with various concentrations of sample extracts, cultures were further incubated for 48 hrs. At the end of the incubation period, cell viability was assayed by measuring the color and fluorescence of MTT and Alamer Blue respectively with a microplate reader. Finally, the 50% reduction in cell number or IC₅₀ was estimated.

Analysis of Total Phenolics:

The total phenolic content of root derived suspension culture of Morinda citrifolia was determined with Folin-Ciocalteu reagent. An aliquot (1 ml) of extract was added to 2.5 ml of 10% dilution of Folin-Ciocalteu reagent and 2 ml of Na₂CO₃(7.5 %, w/v) and incubated at 45ºC for 15 min. The absorbance of samples was measured at 765 nm using a UV-Visible spectrophotometer (Shimadzu UV Mini 1240). Total phenolics were expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g dw).

Analysis of Total Flavonoids:

Total flavonoid content was measured by the aluminium chloride colorimetric assay. An aliquot (1 ml) of extract was added to 10 ml volumetric flask containing 4 ml of distilled water and 0.3 ml 5 % NaNO_2.After 5 min, 0.3 ml 10 % AlCl₃ was added. At 6^th min, 2 ml NaOH (1 M) was added and the total volume was adjusted to 10 ml with distilled water. The solution was mixed well and the absorbance at 510 nm was measured against prepared reagent blank using a UV-Visible spectrophotometer (Shimadzu UV Mini 1240). Total flavonoid content of root suspension culture of Morinda citrifolia was expressed as milligrams of quercetin equivalents per gram of dry weight.

Statistical analysis:

All experiments were conducted in triplicate and statistical analysis was done by using the software Graphpad Prism 5. The data were presented as mean ± SD. Mean differences were considered significant at the P < 0.0001 level.

Results and Discussion:

It has long been studied that the phytochemicals, present in higher plants, play an important role in antioxidant activity and cytotoxicity of these plants. Among those phytochemicals, flavonoids and/or other phenolics are known to possess strong scavenging effect against free radicals such as superoxides and hydroxyl radicals. In the present study, we analyzed root suspension culture of Morinda citrifolia for possible antioxidant and antiproliferative activities. In view of the therapeutic potential of flavonoids and phenolic compounds, we have also quantified the content of these useful classes of secondary metabolites in the Morinda citrifolia root culture.

DPPH Assay:

DPPH is a stable nitrogen-centered free radical. The odd electron in the DPPH free radical gives a strong absorption maximum at 517 nm and is purple in color. The color turns from purple to yellow as the molar absorptivity of the DPPH radical at 517 nm reduces from 9660 to 1640 when the odd electron of DPPH radical becomes paired with hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H. The DPPH radical scavenging activity of the root suspension culture extract is shown in fig.1. Morinda citrifolia cell suspension culture extract showed 50% inhibition (IC₅₀) at 92.5 ± 5.65 (n= 3) µg/ml. Glutathione and β-carotene were used as reference compounds. Glutathione was the most efficient by lowest IC₅₀ value, 63.5 ± 2.12 (n= 3) µg/ml while β-carotene was least efficient with the highest IC₅₀ value, 527.5 ± 10.6 (n= 3) µg/ml. Thus, the root suspension culture of Morinda citrifolia showed higher radical scavenging activity than that of β-carotene but less than glutathione.

Fig.1: DPPH radical scavenging activity of root suspension culture extract, glutathione and β-carotene (n = 3).

Reducing Power Assay:

The reducing capacity of test compounds, often measured by quantifying the transformation of Fe³⁺ to Fe²⁺, serve as a significant indicator of their potential antioxidant potential¹⁷. The reducing power of the methanol extract of Morinda citrifolia root suspension culture is shown in fig.2. Ascorbic acid was used as reference compounds. The reducing power of the extract along with the reference compound (ascorbic acid) was found to increase with rising concentrations. It was evident that, the root suspension culture extract displayed by order of magnitude more reducing potential than ascorbic acid.

Fig.2: Representative result showing the reducing power activity of the root suspension culture extract of Morinda citrifolia and Ascorbic acid (n = 3).

Cytotoxicity of cell suspension culture extract:

Cytotoxicity of cell culture extract of Morinda citrifolia was evaluated on mouse melanoma cells (B16) and human breast cancer cells (MCF-7). When cells were treated for 48 hrs with various concentrations of root suspension culture extract, the relative cell survival progressively decreased in a dose-dependent manner. The suspension culture extract of Morinda citrifolia displayed toxicity with IC₅₀ < 1 mg/ml and they showed significant cytotoxicity with IC₅₀ 295 ± 14.14 (n= 3) µg/ml and 472.5 ± 17.67 (n= 3) µg/ml on B16 and MCF-7 cells respectively as shown in fig 3.

Fig.3. Cytotoxicity shown by root suspension culture extract of Morinda citrifolia on B16 (mouse melanoma) and MCF-7 (human breast cancer) cells (n = 4).

Total phenolics and flavonoids:

Total phenolics and flavonoids were measured by Folin-Ciocalteau reagent and aluminium chloride colorimetric assays respectively. Since these classes of natural products are known to have antioxidant activities, it is likely that the activity of Morinda citrifolia root culture extracts is due to the presence of these compounds. This activity is believed to be mainly due to their redox properties which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides¹⁸.

In root suspension culture extract, the total phenolics were found to be 22.4 ± 0.28 mg/g (n=3) dry weight whereas total flavonoids were found to be 218 ± 6.26 (n=3) mg/g dry weight. The results strongly showed that the culture is rich in flavonoids and phenolics. Hence, flavonoids and phenolics in Morinda citrifolia mainly might contribute to the antioxidant as well as antiproliferative activities of this plant.

Oxidative stress induced by reactive oxygen species (ROS) is now recognized to be a prominent feature of many acute and chronic diseases and even cancer and leukemia^19-22. It has also been implicated in the pathology of many diseases and conditions including diabetes, cardiovascular diseases and inflammatory conditions cancer and ageing²³. Antioxidants may offer resistance against the oxidative stress by scavenging free radicals, inhibiting the oxidation of biological molecules and/or by many other mechanisms. Such effects are attributed to the beneficial effects of antioxidants in disease prevention²⁴including cancer²⁵.

Phenolic compounds present in the plants are good electron donors and scavengers of active oxygen species²⁶. They may accelerate the conversion of hydrogen peroxide into H₂O and their presence in plants often correlate with their free radical scavenging properties^27,28. In addition to free radical scavenging, the antioxidant effect of phenolic compounds is also associated with their ability to chelate and metals²⁹. Flavonoids are other group of free radical scavenger natural products with diverse biological activities. Among the well studied biological effects of flavonoids, reported inhibitory effect on cancer initiation, cancer development and tumor invasion. Flavonoids appear to have ideal structural chemistry for free radical-scavenging activities and have been shown to be more effective antioxidants in vitro than vitamins E and C on a molar basis³⁰.

CONCLUSION:

The detection of high level of flavonoids and phenolics in the root suspension culture of Morinda citrifolia means that they may be responsible for the observed potent antioxidant and antiproliferative activity of the sample. Further research is required to isolate and identify the active principles and also validate the therapeutic potential of present findings.

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Received on 09.05.2010 Modified on 27.05.2010

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1189-1193