Various parts of Chrysanthemum are used as anti-inflammatory and blood purifier
medicines. The inflorescence or bud of Chrysanthemum indicum has long
been used as a Chinese traditional medicine, particularly for the treatment
of inflammation, hypertension and respiratory diseases. Earlier studies have
report that Chrysanthemum indicum possesses anti bacterial, anti virus,
anti oxidant, anti-inflammatory, immunomodulatory (Wang
et al., 2000) and anti-gout properties at high xanthine oxidase inhibition
activity (Nguyen et al., 2004). However, C.
indicum needs a suitable growing area such as highland for them to give
flowers consistently throughout the year, thus, most of the lowlands in Malaysia
are not suitable for this activity.
In recent years, various plant cell-culture systems have been exploited for
the enhancement of high value metabolites. However, looking at the low production
of this secondary metabolite and scarcity of the source material, it is suggested
that it is not so viable. In view of this, studies on the enhancement of xanthine
oxidase inhibitor using plant-cell and tissue-culture together with elicitor
have been carried out. Numerous approaches have been developed to enhance the
productivity of plant-cell and tissue-culture such as medium optimization, cell
line selection, cell immobilization, precursor addition, elicitation, genetic
transformation, organ or hairy root cultures, metabolic engineering and integrated
bioreactor engineering. Elicitation was most successful in cell-culture of various
plant species in order to boost the production of secondary metabolites in plants
(Abdullah et al., 2005).
Elicitation is a method adopted for the purpose of enhancing the secondary
metabolite production. It is defined as the induction of secondary metabolite
production by molecules or elicitor treatment (Singh, 1999).
The recent development of elicitation has opened a new avenue in the area of
production of secondary metabolites (DiCosmo and Misawa,
1985). The elicitors prepared from Rhizoctonia solani, improved the
solavetivone production in Hyoscyamus muticus (Ramakrishna
et al., 1993) and Asperigillus flavus mycelial extract
elicited anthocyanin content in Daucus carota cell-culture (Rajendran
et al., 1994). It has also been reported that increased levels of
kalopanaxsaponin in leaves of Nigella sativa by methyl jasmonate (Scholz
et al., 2009) and chitosan induced anthraquinones production in Rubia
tinctorum suspension cultures (Perassolo et al.,
2008). Use of elicitors, which are not specific to the species or an inappropriate
production medium, can cause ineffective elicitation. Funk
and Brodelius (1990) failed to induce the phenylpropanoid pathway in cell-suspensions
of Vanilla planifolia by using yeast elicitor.
Thus, successful application of elicitation is a challenging task and requires an intensive and prolonged trial and error procedure. Despite the importance of elicitation for enhancing accumulation of secondary products and for a better understanding of their biosynthesis and regulation, such studies have never been carried out on C. indicum cultures. Therefore, in order to induce more xanthine oxidase inhibitor activity, different kinds of inducers (elicitor) have been applied in a stipulated amount and at the suitable time.
MATERIALS AND METHODS
This study was carried out in Faculty of Engineering, International Islamic University Malaysia in the year 2007.
Callus and elicitors: Flowers of C. indicum were obtained from C. indicum farm in Cameron Highland, Pahang, Malaysia, latitude: 4 29 00 and longitude: 101 27 00. Chitosan extracted from crab-shell [Mr ~ 400000] was purchased from SIGMA, Malaysia, yeast extract from Difco and A. niger strain A103 from lab stock of the Department of Biotechnology Engineering, Faculty of Engineering, International Islamic University Malaysia.
Chrysanthemum indicum callus induction: Chrysanthemum indicum callus was induced by culturing surface sterilized flower explants of C. indicum on MS (Murashige and Skoog) semisolid media (8 g L-1 plant agar, Phytotech) supplemented with 0.5 mg L-1 6-BA(6-benzylaminopurine) and 2.0 mg L-1 NAA (naphthalene acetic acid). They were cultured for 3 weeks in dark conditions at 25°C.
Chrysanthemum indicum cell suspension culture preparation and establishment of the growth curve: Initially, about 1.0 g fresh C. indicum callus, grown on MS semisolid media, were cut into small pieces and sub cultured on the same fresh MS media for proliferation. The C. indicum callus, weighing about 0.5 g, was then gently cut using forceps and blades into 20-30 small pieces and transferred into 100 mL MS liquid media. Three replicated samples were then prepared. The Erlenmeyer flask containing cell suspension culture of C. indicum callus was cultured on a rotary shaker set at 125 rpm and 25°C temperature.
The C. indicum cell suspension culture was then sub-cultured every week. Once the suspension culture become established having finely dispersed cell clusters and aggregates, a dilution ratio of 1:10 old culture to fresh medium could be possible on 7-10 days basis to maintain the cell-line. The well established cell suspensions were used as inoculums for the elicitor treatment.
Experimental design: Design of the experiment (DOE) and statistical
analysis were done using statistical software, STATISTICA 6.0. The optimization
study was conducted using general full factorial design with two factors and
three levels (Table 1). The numbers of runs were 9 and average
of triplicate readings for all the runs was recorded for accuracy. The selected
factors were elicitor treatment time (day) and concentration of elicitors (g
L-1). Same DOE was used for all selected elicitors of chitosan, A.
niger and yeast. The xanthine oxidase inhibition percentage was taken as
the dependent variable or response (Y). A second order polynomial equation was
then fitted to the data by multiple regression procedure. This resulted in an
empirical model that related the response measured in the independent variables
to the experiment. For a two-factor system, the model equation is:
where, Y is the XO inhibitor activity (%), β0, intercept;
β1, β2, linear coefficients; β11,
β22, squared coefficient and β12, interaction
Elicitor preparation and treatment: Chitosan, A. niger and yeast
were bought and prepared beforehand. Percentage inhibition for control samples
were also recorded for comparing the improvement in XO inhibition activity after
the elicitor treatment (Table 2).
||Design of experiment for elicitor treatment using different
||Percentage inhibition for control samples
|*Sample for control 1-3 was taken from cell suspension culture
of day 1, 3 and 6, which are not treated with elicitor
Chitosan was prepared as suggested by Popp et al.
(1997). Chitosan was dissolved in 5% (v/v) 1N hydrochloric acid (HCl) through
gentle heating and continuous stirring. pH was adjusted to 5 with 1N sodium
hydroxide (NaOH) and the final concentration was adjusted to 10 mg mL-1.
The solution was stirred to dissolve chitosan and then autoclaved for 15 min
at 121°C. Finally, the solution was kept at 4°C prior to use. The concentrations
for elicitation process prepared were 0.05, 0.10 and 0.25 g L-1 as
stated in Table 3.
The medium for growing A. niger spores (30 g L-1 glucose,
2.5 g L-1 NH4NO3, 1.0 g L-1 KH2PO4,
0.25 g L-1 MgSO4.7H2O, 0.05 g L-1
ZnSO4.7H2O) was prepared and sterilized by autoclaving
for 15 min at 121°C. Inoculums were prepared (spore suspension) according
to the method suggested by Jamal et al. (2005).
Cultures grown at 32°C for 7 days was transferred into Erlenmeyer flask
(250 mL) containing 100 mL of sterile distilled water. The flasks were shaken
in a rotary shaker at 150 rpm for 24 h. The suspended fungal cultures were filtered
using Whatmann No. 1 filter paper. Finally the filtrate was used as inoculums.
For the preparation of the fungal elicitors, about 15 mL of A. niger
inoculums were transferred into 500 mL flask containing 150 mL medium and was
then cultured at 30°C at 150 rpm on a rotary shaker. The A. niger
was harvested after 7 days of cultivation using Whatmann No. 1 filter paper.
It was then dried in an oven at 90°C for 24 h and then grinded using mortar
The dried A. niger powder was measured and dissolved in distilled water to obtain concentration of 10 g L-1. The mixture was then autoclaved at 121°C for 15 min before being added to the plant cell-culture. Finally, A. niger was prepared according to the desired concentration as shown in Table 4.
Ten gram yeast was dissolved in 100 mL of double distilled water and ethanol was added up to 80% (v/v) and kept at 4°C for 3 days for precipitation. The supernatant was then decanted and the precipitate was dissolved in 100 mL of double distilled water and then steam sterilized using autoclave for 15 min at 121°C. After sterilization, the YE elicitor was stored in a chiller before using it for elicitor treatment. The concentrations for elicitation process selected were 0.5, 1.0 and 1.5 g L-1 as stated in Table 5.
About 100 mL C. indicum suspension cells grown in 250 mL Erlenmeyer
flasks were treated with three different concentrations of chitosan, A. niger
and YE. Varying concentrations of elicitors were added to the cultures during
day 1 (lag phase), day 3 (log phase) and day 6 (stationery phase) of growth
as stated in Table 2, 4 and 6.
After the elicitation, the cultures were maintained at 25°C in the dark
for four days. Thereafter, the cells were filtered using Whatmann No. 93 filter
paper. All experiments were carried out in triplicate and an average was taken
in order to get an accurate result. Control sample was not elicited with any
||Experimental and predicted percentage of XO inhibition when
treated with chitosan elicitor
||Experimental and predicted percentage of XO inhibition when
treated with A. niger elicitor
||Experimental and predicted percentages of XO inhibition when
treated with yeast elicitor
||ANOVA for the selected quadratic model (Chitosan)
The flirted cells were weighed after drying (at 60°C for 24 h) and grinding.
The active compounds were extracted from the dried cells. The samples were kept
in tubes and 1.5 mL of distilled water was added to each samples. The extraction
process was carried out using water bath at 30°C for 16 h. The extracts
were then diluted with phosphate buffer to make a concentration of 100 μg
mL-1 (test solution).
XO inhibitor activity measurement: The mixture was consisted of 0.3
mL of 0.1 M phosphate buffer (pH 7.5), 0.1 mL of test solution (100 μg
mL-1), 0.1 mL distilled water and 0.1 mL of 0.12 U mL-1
xanthine oxidase. The mixture was incubated at 25°C for 15 min and after
that, the reaction was initiated by the addition of 0.2 mL of 150 mM xanthine
substrate solution. The test mixture was incubated again at 25°C for 30
min. The reaction was stopped by adding 0.2 mL 1 N HCl. The uric acid produced
was monitored at 290 nm using a UV-spectrophotometer. The values obtained were
the mean of the three replications of the sample. Allopurinol (100 μg mL-1)
was used as positive control. XO inhibitory activity was expressed as the percentage
inhibition of XO in the above assay system, calculated as:
where, A and B are the activities of the enzyme without and with test material.
RESULTS AND DISCUSSION
Effect of chitosan, A. niger and yeast extract on XO inhibitor activity:
Data obtained from this experiment (Table 1-7)
shows that chitosan is a potential elicitor that can enhance XO inhibitor activity
compared to other elicitors tested.
||Results of regression analysis of the full factorial design
|S = 20.06; R2 = 71.7%; R2(adj) = 24.5%
It gave inhibition percentage of 61.818% (Table 3) as compared
to control (42.424% in Table 2) inhibition and this has been
1.457 fold higher than the control sample (Table 3) when treated
using a concentration of 0.1 g L-1 on day 1. Although many other
finding showed that elicitation is efficient at late log phase but our finding
was contradict. This might suggests that the elicitation stress alone is not
sufficient to enhance the productivity. It needed the inoculation stress together
with elicitation stress to enhance the productivity of XOI in C. indicum
cell suspensione (Lu et al., 2001). However,
this percentage is still quite low as compared to allopurinol (positive control)
percentage of inhibition. Nevertheless, as compared to the intact plant extracts
(62.500% inhibition, Table 2), the activity is nearly the
same. Moreover, from the results obtained, it can be concluded that elicitation
using chitosan has fairly near to the value of intact plant of C. indicum.
From the study done by Kong et al. (2000), it
is evident that ethanol extracts of C. indicum has inhibited 95% XO using
the same concentration (100 μg mL-1), whereby water extracts
can only inhibit 68% XO. This value is closer close to the value obtained from
this study, which is 62.5% for intact plant of C. indicum at the same
concentration. It can, thus, be concluded that the low percentage inhibition
obtained for this study was due to water extraction and a higher percentage
might be obtained if other type of solvent system was used.
The highest inhibition of A. niger was 62.121% (Table
5) and 60.606% for yeast (Table 6), but the treatment
time was at day 3, which is much longer than chitosan treatment. The results
prove that treatment with elicitor especially chitosan can be used as a better
alternative to overcome the problem of lower yield in cell suspension culture
system. Some encouraging results were also observed by Zhang
et al. (2007), when the Taxus chinensis cell suspension culture
adapted to chitosan gave 3.2 fold yields and proved that treatment with chitosan
is an effective strategy for improving yield.
Chitosan is the deacetylated form of chitin, which is the main component of
the cell-walls of some fungal species and of the exoskeletons of insects and
crustaceans, being the second most abundantly available natural polysaccharide
on the earth, just next to cellulose. Consequently, chitosan has been widely
applied as a potent elicitor in plant cell suspension cultures to enhance secondary
metabolite production (Kim et al., 1997). Chitosan,
as an effective oligosaccharide elicitor, has also recently been proved to significantly
improve accumulation of secondary metabolites in other plant cell cultures (Kim
et al., 1997; Komariah et al., 2002).
Addition of chitosan elicitor dramatically stimulated PeGs (phenylethanoid glycosides)
biosynthesis in C. deserticola cell-culture, when the stimulation was
associated with PAL (L-phenylalanine ammonia-lyase), the first key enzyme in
PEGs biosynthesis (Cheng et al., 2005). Optimum
incubation time was 48 h for obtaining the highest level of metabolite with
chitosan and A. niger (Komariah et al.,
However, from all the results obtained (Table 3-5),
chitosan gave the highest experimental value. The data obtained was further
analyzed and statistical optimization was done using Statistica software.
Statistical analysis results on elicitation: The regression equation, analysis of result, construction of surface and contour plots were obtained using statistical software, which eventually assisted in determining the optimum treatment time as well as optimum concentration of elicitor to obtain the highest inhibition activity of XO. The statistical optimization predicted the highest inhibition activity of 60.975% at 1.25 day or 30 h culture time using a concentration of 0.16 g L-1 of chitosan.
The fungal elicitor, Aspergillus niger, demonstrated the maximum inhibition at its highest concentration of elicitor on day 1 of cultivation. The percentage inhibition of xanthine oxidase increased by 1.441 fold compared to control sample. However, the predicted value obtained by the software was 0.15 g L-1 of elicitor concentration and the highest inhibition of XO was obtained on 3rd day. The predicted value was 67.435% and the experimental value obtained was 53.333%. It can be noticed that the difference between the predicted and actual value was quite high. On the other hand, inhibitory effects were quite low on day 6 of cultivation, except for 0.15 g L-1 concentration. Compared to sample from intact plant, it can be concluded that the value attained by inhibition of XO by A. niger is closer to the value of intact plant of C. indicum. However, fold increment was still low as compared to chitosan elicitation.
The YE elicitor, induced 1.379 fold percentage inhibition of xanthine oxidase
activity over control sample using 1.5 g L-1 concentration on day
3 of elicitation (Table 5), which was the highest inhibition
attained by YE. The predicted value of highest percentage inhibition of XO (55.005%)
obtained after analysis by the software was 0.15 g L-1 of concentration
and elicitation time of 6 days, which is relatively undesirable considering
cost and time consumption (Table 5). Perhaps for this reason,
Repka (2001) has suggested that it was possible that
different elicitors induced different parts of the defense response. Others
may have synergistic effects on the same pathway. Nevertheless, the host signal
molecules and the mechanisms underlying both elicitor perception at the plant
cell surface and subsequent intracellular transmission of this signal to target
sites are still not fully understood. Therefore, it is hard to explain the underlying
mechanism inhibition obtained by A. niger, chitosan and YE.
Regression analysis was performed to fit the response function with the experimental
data. In this study, the coefficient of variation obtained were: R2
= 0.71 for chitosan, R2 = 0.76 for A. niger and R2
= 0.81 for YE, indicating a relatively high correlation between the experimentally
observed and predicted values. It indicates the degree of precision with which
the secondary metabolite (xanthine oxidase inhibitor) production is attributed
to the independent variables, treatment time and elicitor concentration. Results
clearly indicate that 76.7, 81.7 and 71.7% of the variation in inhibition activities
elicited by A. niger, YE and chitosan respectively is explained by the
model. The polynomial regression Eq. 2-4
were obtained on XO percentage inhibition using chitosan, A. niger, YE,
respectively. The experimental and predicted values for all elicitors are given
in Table 3-5:
The corresponding Analysis of Variance (ANOVA) for chitosan is presented in
Table 6. The computed F-value (1.52) indicates the significance
of the model at high confidence level. The probability p-value was also relatively
low [(pmodel > F) = 0.388), which indicates that the model has
significant explanatory power or the model is good.
Based on the regression analysis for the elicitors, the t-distribution and
the corresponding p-values of the variables along with the second order polynomial
coefficient were evaluated (Table 7). The significance of
each factor was determined by these values because the pattern of interaction
between the factors is indicated by these coefficients. The large magnitude
of t-test and small p-values (less than 0.05) indicate the high significance
of corresponding coefficient. The variables with low probability value contribute
to the model; whereas, variables with high values can be eliminated from the
model. In the Table 7, it can be observed that the chitosan
concentration is the most significant contributing factor. This indicates that
it can act as a limiting factor or a nutrient and little variation in its concentration
will alter the percentage inhibition of XO.
Surface response and contour plot: The surface response analysis (Fig. 1) has estimated the optimum inhibition of xanthine oxidase over independent variables, treatment time (X1) and elicitor concentration (X2). The main objective of response surface is to determine optimum values of the variables efficiently in order to obtain maximized response. Each contour curve represents an infinitive number of combinations of two-test variables. The highest predicted value indicated by surface confined in the smallest ellipse in the contour diagram. Elliptical contours were obtained when there was a perfect interaction between independent variables.
The red elliptical shades from contour plot analysis (Fig. 2)
in the middle of the quadratic curve characterize the optimum area of interaction
between the two parameters to show the maximum percentage inhibition of XO.
||Surface plot for percentage inhibition of XO as a function
of chitosan concentration and treatment time
||Contour plot for percentage inhibition of XO as a function
of chitosan concentration and treatment time
Thus, the maximum percentage inhibition of XO that can be achieved is 60.975%
when elicited at day 1.25 or 30 h at concentration of 0.16g L-1 of
In conclusion, chitosan is the most suitable elicitor for XO inhibitor production
from C. indicum. The optimum concentration of chitosan required is 0.16
g L-1 and the optimum time for elicitor treatment is day 1.25 or
30 h of cultivation to enhance percentage inhibition of XO. This conclusion
was drawn on the basis of the recorded data showing an increase of 1.457 fold
inhibition activity compared to the control sample after being elicited by chitosan
at the optimum conditions. A. niger and YE also exhibit significant effect
in inducing enhancement of XO inhibitor activity. However, chitosan is the best
elicitor and has successfully enhanced the inhibition activity.
We would like to thank International Islamic University Malaysia for providing use budget for this project.