Herbal and natural plants have been used as medicine in centuries in many cultures
in the world. Scientists and medicinal professionals have shown great interests
in doing research in this field recently as they are acknowledged the health
benefits of the plants. Moreover, plants are potential sources of natural antioxidants.
They produce a very imposing group of antioxidant compounds which inclusive
of carotenoids, flavonoids, cinnamic acids, benzoic acids, folic acid, ascorbic
acid, tocopherols and tocotrienols (Hollman, 2001).
These antioxidant compounds are important and beneficial for food protection
as well as a defense mechanism of living cells against oxidative damage (Vimala
and Adenan, 1999).
Morinda citrifolia L., the Mengkudu, is one of the important traditional
Polynesian medicinal plants that have been used for over 2000 years. Known commercially
in Malaysia as Mengkudu, it is also referred as Cheese fruit, Ba Ji Tian, Nhau,
Indian Mulberry throughout the world. Mengkudu is a native plant found around
Southeast Asia to Australia and is cultivated in India, Polynesia, the Caribbean,
Central and Northern South America. Mengkudu is noted for its wide range of
usage as a medicinal plant or as an edible food source. The young leaves of
this plant can be cooked as vegetables and even the dried leaves or fruits are
used to make infusions and teas. Besides that, the roots and bark can be turn
into dyes, while the trunks part can be use as firewood and tools (Yanine
et al., 2006).
Mengkudu has recently been the object of many claims concerning its nutraceutical
properties. It has been recognized as a plant that contains a broad range of
therapeutic effects such as antioxidant, antibacterial, antiviral, antifungal,
antitumor, anti-inflammatory and immune enhancing effects (Wang
et al., 2002). Various publications have shown that mengkudu can
be used to relieve different kinds of illnesses including arthritis, diabetes,
high blood pressure muscle aches and pains, blood vessel problems and cancers.
Two clinical studies reported a relief of arthritis and diabetes associated
with mengkudu consumption (Elkins, 1998; Solomon,
1999): the observed beneficial effects may result from certain compounds
such as scopoletin, nitric oxide, alkaloids and sterols and also to the antioxidant
potential of Mengkudu. Therefore, consumption of this fruit is currently high,
not only in the producing countries but also in the United States, Japan and
Microencapsulation technique is an effective way and widely used to protect
drug or food ingredients against deterioration and volatile losses. The protective
mechanism is to form a membrane wall to enclose droplets or particles of the
encapsulated material. There are currently a variety of means available in regards
of preparing microparticles. The practice of each method is depending on the
types of desired microparticles. It can include the properties of the wall structures,
sizes, shape quantity and quality of final product. Another factor that often
comes into consideration is cost of operation, practicality of the process and
overall time frame. Of all the means, fluid bed coating, centrifugal extrusion
and spray drying are the major commercial processes usually used in terms of
practicality and product volume. However, in food and drug industry, spray drying
is still favoring the most. It is due to its continuous production and easiness
of industrialization (Shu et al., 2008). Nevertheless,
microencapsulation of Morinda citrifolia L. extracts by spray drying
still has not been reported.
For microencapsulation by spray drying, carrageenan is a good choice as wall
material due to its pseudoplastic properties. These properties will allow it
to act as plasticizer, promoting the formation of spherical and smooth-surfaced
microcapsules, enhancing adhesion force between wall and core materials (Shu
et al., 2008). Moreover, it has good properties of emulsification,
edibility and biodegradation. Carrageenan is also one of polysaccharides, which
can be used as excipient in drug industry.
In this study, a spray drying method is developed for preparation of Morinda citrifolia L. mirocapsules using carrageenan as wall material and to investigate the effect of different temperatures on the antioxidant content and activity of the powders.
MATERIALS AND METHODS
Materials: Mengkudu fruit was obtained from various places around Kota
Kinabalu, sabah, Malaysia. The fruits are obtained at unripe stage, tap washed
and separated or the seed and pulp. The pulp is sun dried and further oven dried
until constant weight is obtained. Afterwards, the dried pulp is pulverized
and kept for storage at 40°C.
Extraction of mengkudu: Pulverized antioxidant sample of Mengkudu were extracted using ethyl acetate using 10% solvent concentration. One hundred gram of blended powder was mixed with 1000 mL of ethyl acetate inside a conical flask and left on shaking incubator for 3 days at the temperature of 37°C. The solution was filtered and the filtrate portion was kept at 4°C for further study.
Preparation of emulsion and spray drying: Carrageenan solution was set to 0.5% of concentration. About 0.5 g of carrageenan powder was added into 100 mL of distilled water. During the addition of carrageenan powder, the distilled water in the beaker was stirred and heated up to 80°C in order to obtain a well-mixed solution.
The emulsion of Mengkudu extract and carrageenan was set to 400 mL with selected ratio based on Table 1. The resulting emulsions then were vigorously stirred before fed into a lab plant spray dryer SD05 with the main chamber 215 mm in diameter and height of 500 mm. The spray drier were operated at 5 different temperatures (Table 1).
The pump speed is set to -5.83 to 210 mL h-1, air flowrate of -114 to 69 m3 h-1 and compressor air pressure of 1.2 bar.
Extraction of antioxidant from powder: Two hundred milligram of spray dried powder was carefully weighed and mixed with 2 mL of ethanol and 0.5 mL of distilled water. The solution was then agitated using a vortex for 1 min and centrifuged at 5000 rpm for 10 min. The supernatant was collected as Mengkudu powder extract and kept at 4°C for further analysis.
DPPH Radical Scavenging Activity (RSA): The effect of the microcapsules
extract as an antioxidant source was estimated using free-radical-scavenging
effect on the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical with slight modification
(Alothman et al., 2008). An aliquot of 200 μL-1
powder extract was mixed with 4 mL of 25 mg L-1 methanolic solution
||EY (%), DPPH RSA, Total phenolic content (TPC) and flavonoid
content of microcapsules obtained at different spray-drying condition
The result solution was and left under dark condition for 60 min. Afterwards, the decreased absorbance of the DPPH solution was evenly mixed
measured at 515 nm against blank methanol with Perkin Elmer UV-VIS lambda 25
spectrophotometer. Results were expressed as percentage of inhibition of the
DPPH according to Eq. 1:
where, DPPH RSA is the radical scavenging activity of antioxidant, Abs control is the absorbance of DPPH solution without extracts an abs sample is the absorbance of dpph with sample taken after 60 min.
Total phenolic content: The procedure is based on Folin-Ciocalteau assay,
developed by Singleton and Rossi (1965) with some modification
(Singleton and Rossi, 1965). 1 mL of Folin-Ciocalteau
reagent (1:9:Folin Ciocalteau:distilled water) and 200 μL of sample was
mixed in a tube and left for 5 min at room temperature. After that, 800 μL
of sodium carbonate solution was added; the mixture was then homogenized and
left at room temperature for 2 h. The total phenolic content was further determined
using Perkin-Elmer UV-VIS lambda 25 spectrophotometer at 765 nm. Tannic acid
was used as the standard. The value of phenolic content were expressed in mg
of tannic acid equivalents (TAE)/100 g of powder based on the equation y = 0.007x
(r2 = 0.997).
Total flavonoid content: Total Flavonoid (TF) contents of the Mengkudu
extracts were determined according to the colorimetric assay developed by Zhishen
et al. (1999). One milliliter of powder extract was added with 0.3
mL of (5% w/v) NaNO2. 0.3 mL of (10% w/v) AlCl3 was added
after 5 min, followed by 2 mL of NaOH (1 M) 1 at 6th min. The mixture was shaken
vigorously and the absorbance of the mixture is read at 510 nm. The results
were expressed as mg Catechin Equivalents (CE)/100 g of powder. A calibration
curve was prepared using a standard solution of catechin (20 to 100 mg L-1,
r2 = 0.996).
Encapsulation yield: The Encapsulation Yield (EY) was calculated using
Eq. 2. It was expressed as the ratio of the mass of microcapsules
obtained after spray drying and the mass of initial substances added (Shu
et al., 2008):
Particle size distribution: The size distribution of resulting powder was determined by a laser scattering particle size distribution analyzer instrument (Model LA-300, Horiba instruments Inc).
Scanning Electron Microscopy (SEM): The powder samples were analyzed in a SEM, HITACHI Model S-3700 N (Hitachi High Tech Science Systems Corporation, Japan) in order to examine the external appearance of the particles. The encapsulated samples were fixed in stubs on an adhesive carbon paper. The images of the specimen were obtained at an accelerating voltage of 5 KV and taken at different magnifications ranging 2500 to 7000 SE as indicated on the images.
RESULTS AND DISCUSSION
Antioxidant activity: In this study, the antioxidative activities of
spray dried Mengkudu powder were quantified through the effect of free radical
of DPPH. Table 1 shows the percentage of radical scavenging
activity for DPPH at selected parameter. In this study, Mcore/Mwall
is defined as the volume ratio of Mengkudu extracted sample against wall material
(0.5% carrageenan). Among the Mcore/Mwall ratio, it shows
a higher significant percentage of DPPH inhibition at ratio of 1/2. As the ratio
of Mcore/Mwall decrease from 1/4 to 1/6, the values also
decreasing but only on slight changes. At different drying temperature, the
ratio was set constant at 1/4 and can be compare using runs 2, 4 and 5 (Fig.
1a). The highest antioxidant activity was measured at drying temperature
of 130°C, followed by 110 and 150°C (Fig. 1b). During
the process, the drying temperature must be optimized so that the quantity of
the heat is sufficient to produce microparticles and in the same time retain
a high content of antioxidant. Drying at a higher temperature will lead to degradation
of antioxidative compound and also possibility of losses of volatile substances
(Georgettia et al., 2008).
However, the temperature effect on the relative antioxidant of the extracted
sample may be related to differences in stability or to differences in antioxidant
potencies at various temperatures (Hove and Hove, 1944).
Flavonoid and total phenolic content: Flavonoids and Total Phenolic
Content (TPC) of powdered samples are given in Table 1. Runs
1, 2 and 3 shows results for powder dried at fixed drying temperature of 150°C,
the highest total phenolic content (mg of TAE/100 g of powder) were observed
at ratio Mcore/Mwall of 1/2 with 197 mg of TAE.
Free Radical Scavenging activity of the spray dried Mengkudu
powder: (a) Constant Mcore/Mwall ratio of 1/4 and (b) Constant temperature
ratio of 1/4 and 1/6, the TPC values only slightly varied at 77.5 and 81.43
mg of TAE, respectively. Flavonoid content (mg of CE/100 g of powder) shows the same trend obtained
for TPC analysis. The content of flavonoid was only significantly high when
dried at Mcore/Mwall of 1/2.
At fixed drying ratio Mcore/Mwall of 1/4, the highest
TPC value was achieved at 130°C. TPC shows the lowest content when dried
at a higher temperature. At this condition, there are possible losses or degradation
of certain types of phenolic compounds (Amin et al.,
2006). The highest flavonoid obtained at 120°C with 260 of mg CE, followed
by drying at 110°C. From the results obtained by spray drying of Mengkudu,
it can be concluded that DPPH RSA have an excellent correlation with TPC (r2
= 0.848) and flavonoid (r2 = 0.855). It had been reported that the
antioxidant activity of plant materials is well correlated with the content
of their phenolic group with result found by Miliauskas et
al. (2004) during RSA analysis of plant extract.
The Encapsulation Yield (EY): In the experiment, it is obvious that
temperature and ratio of Mcore/Mwall have a significant
effect on the encapsulation yield of resultant microcapsules. Based on Table
1, comparing at temperature drying rate ranged from 110 to 150°C, the
highest yield was obtained at the highest temperature.
||Mean diameter of powder produce at different Mcore/Mwall
ratio, at temperature of 150°C
||Mean diameter of powder produce at different temperatures,
with Mcore/Mwall of 1/4
At lower temperature, the quantity of the heat is not sufficient to dry the
product and thus there will be a quantity of water exists in the product. The
powder with more water content will easily stuck on the chamber wall and lead
to a low EY (Shu et al., 2008).
The EY will increase further with temperature increment until an optimum point
is achieved before decreasing, as observed by Shu et
al. (2008). This is because at high temperature, the balance between
the rate of water evaporation and film-formation may break leading to broken
microcapsules that will accumulate at the chamber wall and further decreasing
Decreasing the Mcore/Mwall from 1/2 to 1/8 while keeping
other parameters unchanged, at first the EY increased but then decreased when
the ratio reached 1/4. Such negative effect happened probably due to feed viscosity
which exponentially increased with decreasing ratio of Mcore/Mwall.
A greater feed viscosity can cause more solids to paste in the main chamber
wall due to the higher amount of solids in contact with the wall, therefore,
reducing the EY (Tonon et al., 2008). The result
is in agreement with results published by other researcher that stated Mcore/Mwall
= 1/4 was the optimal choice for effective microencapsulation yield (Shu
et al., 2008). Scanning electron micrographs of spray dried powders
of Mengkudu using carragaeenan as wall material are shown in Fig.
Particle size analysis: Table 2 and 3
show the mean diameter at different temperature and ratio, respectively. According
to Table 3, the resultant particle size increased with the
increasing of temperature. Based on Tonon et al.
(2008), higher temperature is related to a higher swelling of droplets and
further results in faster drying rate that produces larger particles than drying
under slower rates. Higher wall material content also led to the production
of larger particles (Table 2). This correlation maybe due
to the feed viscosity used, which increased exponentially with wall concentration.
The result obtained also in agreement with Keogh et al.
(2003) that observed a linear increasing of particle size with feed viscosity.
Scanning electrom micrographs of spray dried powders of Mengkudu
using carragaeenan as wall material: (a) 150°C, 1/2, (b) 150°C,
1/4, (c) 150°C, 1/6, (d) 110°C, 1/4 and (e) 130°C, 1/4
||Correlation of TPC and TF at: (a) constant Mcore/Mwall ratio
of 1/4 and (b) constant temperature at 150°C
Gharsallaoui et al. (2007) stated that at higher
liquid viscosity, a larger droplet is formed during the atomization process
thus, will produced larger particle size.
SEM: Powders morphology: SEM micrograph of Mengkudu spray-dried at various
parameters (Fig. 2a-e) confirmed that the
particles powders is in the range of micro size (1-20 μm). Most of the
particles had spherical shape and less agglomerated. All of the spray dried
microcapsules containing carrageenan with different antioxidant equivalent appeared
to be a smooth spheres. The presence of coating agent such as carrageenan will
react as plasticizer and is important on promoting the formation of spherical
and smooth surfaced microparticles (Bruschi et al.,
2003). Correlation of TPC and TF is shown in Fig. 3a and
Mengkudu microparticles were prepared by means of spray drying. Carrageenan at different ratio were used to prepare the fine particles and showed significant effect on all the response studied. The main factor affecting the antioxidant content is drying temperature. Drying at higher temperature had dramatically decreased the antioxidant potential of resultant powder. However, it was observed that Mcore/Mwall also plays an important role on determining the highest and lowest possible range for optimized outcome antioxidant content. In respect to powder morphology, increasing the temperature resulted particles with larger sizes and higher yield. While, increasing Mcore/Mwall ratio led to the production of smaller particles, which is related to the increase on feed viscosity.
This research was fully supported by Ministry of Science, Technology and Innovation of Malaysia under the project SCF0049-IND-2007. Author also would like to acknowledge Awang Bono from Universiti Malaysia Sabah for his expertise and knowledge.