Antioxidant Activity of Waterleaf (Talinum triangulare (Jacq.) Willd) Cultivated with Organic Fertilizers during the Rainy Season
ANTIOXIDANT ACTIVITY OF WATERLEAF
(Talinum triangulare (Jacq.) Willd) CULTIVATED WITH
ORGANIC FERTILIZERS DURING THE RAINY SEASON
STELLA ALINNESHIA
DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY
FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
STATEMENT LETTER OF MANUSCRIPT AND SOURCES OF
INFORMATION*
Hereby I genuinely stated that the manuscript entitled Antioxidant Activity of
Waterleaf (Talinum triangulare (Jacq.) Willd) Cultivated with Organic
Fertilizers during the Rainy Season is an authentic work of mine under the
supervision of academic counselors and has never been presented in any forms and
universities. All the information taken and quoted from published or unpublished works
of the writters has been mentioned in texts and attached in the bibliography at the end
of this manuscript.
Hereby I bestow the copyright of my manuscript to the Bogor Agricultural
university.
Bogor, September 2014
Stella Alinneshia
F24100122
ABSTRACT
STELLA ALINNESHIA. Antioxidant Activity of Waterleaf (Talinum triangulare
(Jacq.) Willd) Cultivated with Organic Fertilizers during the Rainy Season.
Supervised by PROF. DR. IR. RIZAL SYARIEF, DESS and DR. IR. DIDAH NUR
FARIDAH, M.Si.
Talinum triangulare (Jacq.) Willd, known as ginseng Jawa or kolesom Jawa
in Indonesia, is one of tropical plants that has been widely used as traditional herbal
medicine and consumed as vegetable in Indonesia. The leaf from the plant contains
bioactive compound identified as flavonoid which can function as antioxidant. The
objective of this research was to measure the antioxidant activity and total phenolic
compound of waterleaf in five different treatments of organic fertilization.
Extraction of the leaf was carried out with the method of ultrasound assisted
extraction/UAE (sample:MeOH=1:10 b/v, t=20 minutes, cold extraction room
temperature). The present research resulted that the different organic fertilizer
treatments significantly influenced the secondary metabolites content e.g. phenolic
content of Talinum triangulare (Jacq.) Willd (p 5.5) according to Munawar (2011).
Potassium content of the soil before and after the application of organic fertilizer
was found to be low (Table 2) since the conversion of potassium from organic
fertilizer to more readily available form of potassium happened slowly forcing
9
plants to take more potassium from the soil. That condition resulted in low
potassium content of the soil at the end of the cultivation.
The availability of nutrients (N, P, and K) was influenced by the ratio of C/N
in the soil as shown in Table 3. The result of C/N ratio in five groups was less than
20. According to Munawar (2011), the ratio of C/N falls below 20 prompts
mineralization in which conversion from organic to inorganic form of nutrient
happen providing the plants with readily available nutrients.
Data obtained of the rainfall during the cultivation of waterleaf categorized
as medium rainfall (BMKG 2005). Rainfall influenced the temperature, erosion of
nutrient in the soil, and secondary metabolite formed. Water shortage generally
induced the formation of secondary metabolite in plant increasing the content of
flavonoid as the natural defense mechanism of the plant (Mualim 2012). High
rainfall eroded the nutrient of the soil possibly lowering the nutrient content in the
soil affecting the content of secondary metabolite formed.
Table 4 Moisture content of fresh and dried sample of waterleaf
Moisture Content of Dried Moisture Content of Fresh
Treatment
Sample (%) wb
Sample (%) wb
Control
7.90
90.82
PK+RP
5.58
90.56
PK+AS
6.56
91.01
RP+AS
8.00
91.47
PK+RP+AS
8.36
91.47
PK: cow manure, RP: rock phosphate, AS: husk ash
Moisture content of both fresh sample and dried powdered sample was
measured by AOAC method resulted in the range of 90 – 91 % (wb) and 5 – 8 %
(wb) respectively (Table 4). The moisture content of waterleaf was reported to be
influenced by frequency of fertilizing and the age of the plant: increasing with the
addition of the plant age (Anna 2010).
Sample Extraction
Extraction methods commonly used for obtaining extractable substances
from plant were hydro-distillation method, maceration, Soxhlet extraction, and
ultrasound extraction. Antioxidant compound from Talinum triangulare (Jacq.)
Willd underwent single extraction by ultrasound assisted extraction (UAE) (t=20
minutes, cold extraction room temperature) based on the method stated by
Velickovic (2006) to extract bioactive component from garden sage Salvia
officinalis L. and Salvia glutinosa L.. As an innovative method of extraction, UAE
was said to be very promising and effective to obtain bioactive substances from the
plant like sage and demonstrated the higher yields of extractable component in
which the time is much shorter than classical maceration. The study also showed
that the extract contained more flavonoids when it was extracted by UAE than
classical maceration. UAE has been widely used to extract bioactive component in
walnut green husk (Tabaraki and Rastgoo 2014), Orthosiphon stamineus (Ho et al.
2014), grape seeds (Ghafoor et al 2009), and winter melon seeds (Bimakr et al.
2013). According to Andarwulan (2010) the leaves of Talinum triangulare (Jacq.)
Willd contained bioactive component known as antioxidant from flavonoids.
10
Kaempferol was found to be dominant (90 %) in Talinum triangulare (Jacq.) Willd
followed by quercetin (Andarwulan et al. 2010). Methanol (MeOH) was used to
extract the antioxidant component from the plant as said in the previous study
conducted by Sandrasari (2008).
Five treatments of sample were single extracted with MeOH
(sample:solvent=1:10 b/v) by UAE for 20 minutes, shorter time required compared
to classical maceration performed in previous research by Sandrasari (2008) in
extracting the antioxidant component from the indigenous vegetables. Velickovic
(2006) stated that the maximum concentration of extractable substances in liquid
extract was reached within 20 minutes of extraction at 40 ± 1 oC. The liquid extracts
of Talinum triangulare (Jacq.) Willd were all dark green in color with different
intensity in each treatments (Figure 2).
Figure 2 (Left-Right) Methanol extract of waterleaf treatment control, PK+RP,
PK+AS, RP+AS, and PK+RP+AS
Analysis of Total Phenolic Compound and Antioxidant Activity
The liquid extracts of antioxidant from five groups were analyzed for its total
phenolic compound based on the Folin-Ciocalteau method previously done by
Shetty et al. (1995). Total phenolic compound was measured by Folin-Ciocalteau
method estimating all flavonoids, anthocyanins, and non-flavonoid phenolic
compounds which present in the extract. Folin Ciocalteau method was based on
oxidation-reduction reaction in which phenolic compounds are oxidized with
simultaneous reduction of phosphotungsten-phosphomolybdate complex in base or
alkaline medium turning the color into blue (Figure 3) that can be measured by
spectrophotometer at 765 nm (Gawron-Gzella et al. 2012). The standard of gallic
acid was used and the result of total phenolic was expressed as milligram equivalent
of gallic acid per gram sample. The absorbance value represented the intensity of
phenolic compound in the sample. Greater amount of phenolic compound gave
higher absorbance value.
The extract of the waterleaf was also tested for its antioxidant activity in
scavenging the radicals by DPPH method. DPPH was a stable free radical dissolves
11
in methanol. DPPH became lighter in color (Figure 3) when an antioxidant
scavenges free radicals by hydrogen donation that can be measured at 517 nm
(Gawron-Gzella 2012). The analysis of reducing power was done by the method of
ferric cyanide reducing power. Antioxidant reduced the complex of ferric cyanide
to Fe2+ ion that could be detected by the formation of Prussian blue color (Figure 3)
measured at 700 nm (Sandrasari 2008). Higher absorbance value indicated greater
reducing power of antioxidant.
Figure 3 (Left-right) Total phenolic assay, DPPH assay, and ferric reducing assay
Total phenolic compound of five different treatments of organic fertilizer
showed variation of the result ranged from 6.00 – 10.76 mg GAE/ g dry sample
(wb) (Table 5). The highest total phenolic was found in PK+RP (10.76 mg GAE/ g
dry sample wb) and the lowest total phenolic was found in PK+AS (6.00 mg GAE/
g dry sample wb). In sequential order the highest to the lowest total phenolic content
was PK+RP, control treatment, PK+RP+AS, RP+AS, and PK+AS. Previous
research conducted by Andarwulan (2010) resulted that the phenolic compound
commonly found in ten indigenous vegetables among them katuk (Sauropus
androgynous (L) Merr), kenikir (Cosmos caudatus H.B.K), kedondong cina
(Polyscias pinnata) was ranged from 0.33 – 0.152 mg GAE/ g fresh weight and the
total phenolic content of waterleaf was 0.489 mg GAE/g fresh weight. Compared
to present research, the total phenolic compound was higher in the present research.
Andarwulan et al. (2010) stated that the phenolic compound in waterleaf was
dominated by kaempferol and very little amount of quercetin. The difference of
total phenolic compound between current and previous research indicated that
different solvent and method of extraction used would result in significant different
amount of both kaempferol and quercetin that could be extracted from the samples.
Ethanol 95 % and classical shaking maceration were used in previous research
while methanol and ultrasound assisted extraction (UAE) were used in current
research. UAE proved to be more effective in extracting the phenolic compound
than classical maceration as previously said by Velickovic et al. (2006) in term of
yielding more extractable component.
The DPPH antioxidant activity was expressed as IC50 value. IC50 value
showed the concentration of antioxidant extract from waterleaf to reduce 50 % of
DPPH radical. Lower concentration indicated stronger antioxidant activity. The
extract that had strongest ability to reduce 50 % of DPPH radical was RP+AS
extract (1170.20 ppm) and the weakest one was PK+AS (2396.54 ppm). The result
in sequential order from the strongest to the weakest based on IC50 value was
RP+AS, control treatment, PK+RP, PK+RP+AS, and PK+AS (Table 5).
12
Table 5 Total phenolic and antioxidant activity
Treatment
Total Phenolic*
IC50 Value**
DPPH
AEAC***
Ferric
Reducing
AEAC**
DPPH
Antioxidant
Activity****
Ferric Reducing
Antioxidant
Activity****
Control
9.75d ± 0.16
1244.12a ± 6.44
16.92c ± 0.12 0.14c ± 0.01
1.74b ± 0.04
0.015c ± 0.000
PK+RP
10.76e ± 0.06
1231.23a ± 12.85
18.65d ± 0.00 0.18e ± 0.01
1.73b ± 0.01
0.018d ± 0.001
PK+AS
6.00a ± 0.13
2349.54b ± 1.17
8.50a ± 0.10
0.01a ± 0.00
1.42a ± 0.02
0.002a ± 0.000
RP+AS
7.36b ± 0.00
1170.20a ± 7.78
15.18b ± 0.09 0.16d ± 0.00
2.06c ± 0.01
0.022e ± 0.000
PK+RP+AS
8.85c ± 0.03
1303.42a ± 265.54
15.24b ± 0.37 0.07b ± 0.00
1.72b ± 0.04
0.008b ± 0.000
PK: cow manure
RP: rock phosphate
AS: husk ash
*mg GAE/g dry sample
**mg/g dry sample
***mg AEAC/g dry sample
****mg AEAC/mg GAE
13
Both DPPH and ferric reducing power antioxidant activity were expressed in
AEAC or Ascorbic acid Equivalent Antioxidant Activity by using vitamin C or
ascorbic acid as the reference standard. The value then calculated in the basis of
one gram of total phenolic to show the antioxidant activity per gram total phenolic
(Table 5). The highest antioxidant activity to scavenge DPPH radical per gram total
phenolic was found in RP+AS group (2.06 ± 0.01 mg AEAC/g GAE). The lowest
was found in PK+AS group (1.42 ± 0.02 mg AEAC/g GAE). Control, PK+RP,
PK+RP+AS treatments followed the order from the highest. While for reducing
power, the group with the strongest reducing power was found in RP+AS (0.022 ±
0.00 mg AEAC/g GAE) followed by PK+RP, control, PK+RP+AS, and PK+AS.
Thus could be inferred that high total phenolic content is not always resulting in
high antioxidant activity, but high ability to scavenge free radical will result in high
reducing power. The highest total phenolic content was found in group PK+RP
(10.76 mg GAE/g dry sample), but its antioxidant was weaker in scavenging DPPH
radical and reducing the ferric (1.73 mg AEAC/g GAE and 0.018 mg AEAC/g
GAE) compared to RP+AS (2.06 mg AEAC/g GAE and 0.022 mg AEAC/g GAE).
Thus could be inferred that the organic fertilizer treatment influenced the secondary
metabolite produced in which the high antioxidant activity resulted from other
components (e.g. vitamin C and carotenoid) rather than only phenolic compounds.
It was found that fertilizer rich in soluble nitrogen (e.g. PK or cow manure) could
decrease the ascorbic acid content since nitrogen supply increased plants’ leaf
density. Carotenoid content was said not being influenced by fertilizer treatments
(Zoran et al. 2014). Compared to other vegetables in previous research, the
antioxidant activity of waterleaf was weaker. Kaempferol existed as the only
dominated flavonoid antioxidant found in waterleaf while beluntas (Pluchea indica
Less.) and kenikir (Cosmos caudatus H.B.K) contained quercetin, miricetin instead
of kaempferol (Andarwulan 2010; Sandrasari 2008). Antioxidant activity of
flavonoid depended on the number and location of phenolic group –OH to
neutralize free radical. Quercetin, miricetin and kaempferol had double bond on C2C3 increasing its ability to scavenge free radical. Structure of 3,4-dihydroxyl
(cathecol in B ring) in those three could act as electron donor for targeted radical
(Amic et al. 2002). The amount of hydroxyl group, however, differed from one to
another (quercetin=5, miricetin=6, kaempferol=4) making kaempferol had less
antioxidant activity compared to quercetin and myricetin resulting that the
antioxidant activity of the waterleaf was weaker among other indigenous vegetables
i.e. beluntas (Pluchea indica Less.) and kenikir (Cosmos caudatus H.B.K).
Table 6 Total flavonoid of the waterleaf in different organic fertilizers (Saleh
2013)
Treatment
Flavonoid (mg QE/g dry weight)
Control
PK + RP
PK + AS
RP + AS
PK + RP + AS
PK: cow manure, RP: rock phosphate, AS: husk ash
10.62a ± 2.8
11.07a ± 3.1
9.10a ± 2.7
8.90a ± 1.2
9.71a ± 1.6
14
Based on ANOVA (Table 5 and appendices), total phenolic value was
significantly different between groups of organic fertilizer (p
(Talinum triangulare (Jacq.) Willd) CULTIVATED WITH
ORGANIC FERTILIZERS DURING THE RAINY SEASON
STELLA ALINNESHIA
DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY
FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
STATEMENT LETTER OF MANUSCRIPT AND SOURCES OF
INFORMATION*
Hereby I genuinely stated that the manuscript entitled Antioxidant Activity of
Waterleaf (Talinum triangulare (Jacq.) Willd) Cultivated with Organic
Fertilizers during the Rainy Season is an authentic work of mine under the
supervision of academic counselors and has never been presented in any forms and
universities. All the information taken and quoted from published or unpublished works
of the writters has been mentioned in texts and attached in the bibliography at the end
of this manuscript.
Hereby I bestow the copyright of my manuscript to the Bogor Agricultural
university.
Bogor, September 2014
Stella Alinneshia
F24100122
ABSTRACT
STELLA ALINNESHIA. Antioxidant Activity of Waterleaf (Talinum triangulare
(Jacq.) Willd) Cultivated with Organic Fertilizers during the Rainy Season.
Supervised by PROF. DR. IR. RIZAL SYARIEF, DESS and DR. IR. DIDAH NUR
FARIDAH, M.Si.
Talinum triangulare (Jacq.) Willd, known as ginseng Jawa or kolesom Jawa
in Indonesia, is one of tropical plants that has been widely used as traditional herbal
medicine and consumed as vegetable in Indonesia. The leaf from the plant contains
bioactive compound identified as flavonoid which can function as antioxidant. The
objective of this research was to measure the antioxidant activity and total phenolic
compound of waterleaf in five different treatments of organic fertilization.
Extraction of the leaf was carried out with the method of ultrasound assisted
extraction/UAE (sample:MeOH=1:10 b/v, t=20 minutes, cold extraction room
temperature). The present research resulted that the different organic fertilizer
treatments significantly influenced the secondary metabolites content e.g. phenolic
content of Talinum triangulare (Jacq.) Willd (p 5.5) according to Munawar (2011).
Potassium content of the soil before and after the application of organic fertilizer
was found to be low (Table 2) since the conversion of potassium from organic
fertilizer to more readily available form of potassium happened slowly forcing
9
plants to take more potassium from the soil. That condition resulted in low
potassium content of the soil at the end of the cultivation.
The availability of nutrients (N, P, and K) was influenced by the ratio of C/N
in the soil as shown in Table 3. The result of C/N ratio in five groups was less than
20. According to Munawar (2011), the ratio of C/N falls below 20 prompts
mineralization in which conversion from organic to inorganic form of nutrient
happen providing the plants with readily available nutrients.
Data obtained of the rainfall during the cultivation of waterleaf categorized
as medium rainfall (BMKG 2005). Rainfall influenced the temperature, erosion of
nutrient in the soil, and secondary metabolite formed. Water shortage generally
induced the formation of secondary metabolite in plant increasing the content of
flavonoid as the natural defense mechanism of the plant (Mualim 2012). High
rainfall eroded the nutrient of the soil possibly lowering the nutrient content in the
soil affecting the content of secondary metabolite formed.
Table 4 Moisture content of fresh and dried sample of waterleaf
Moisture Content of Dried Moisture Content of Fresh
Treatment
Sample (%) wb
Sample (%) wb
Control
7.90
90.82
PK+RP
5.58
90.56
PK+AS
6.56
91.01
RP+AS
8.00
91.47
PK+RP+AS
8.36
91.47
PK: cow manure, RP: rock phosphate, AS: husk ash
Moisture content of both fresh sample and dried powdered sample was
measured by AOAC method resulted in the range of 90 – 91 % (wb) and 5 – 8 %
(wb) respectively (Table 4). The moisture content of waterleaf was reported to be
influenced by frequency of fertilizing and the age of the plant: increasing with the
addition of the plant age (Anna 2010).
Sample Extraction
Extraction methods commonly used for obtaining extractable substances
from plant were hydro-distillation method, maceration, Soxhlet extraction, and
ultrasound extraction. Antioxidant compound from Talinum triangulare (Jacq.)
Willd underwent single extraction by ultrasound assisted extraction (UAE) (t=20
minutes, cold extraction room temperature) based on the method stated by
Velickovic (2006) to extract bioactive component from garden sage Salvia
officinalis L. and Salvia glutinosa L.. As an innovative method of extraction, UAE
was said to be very promising and effective to obtain bioactive substances from the
plant like sage and demonstrated the higher yields of extractable component in
which the time is much shorter than classical maceration. The study also showed
that the extract contained more flavonoids when it was extracted by UAE than
classical maceration. UAE has been widely used to extract bioactive component in
walnut green husk (Tabaraki and Rastgoo 2014), Orthosiphon stamineus (Ho et al.
2014), grape seeds (Ghafoor et al 2009), and winter melon seeds (Bimakr et al.
2013). According to Andarwulan (2010) the leaves of Talinum triangulare (Jacq.)
Willd contained bioactive component known as antioxidant from flavonoids.
10
Kaempferol was found to be dominant (90 %) in Talinum triangulare (Jacq.) Willd
followed by quercetin (Andarwulan et al. 2010). Methanol (MeOH) was used to
extract the antioxidant component from the plant as said in the previous study
conducted by Sandrasari (2008).
Five treatments of sample were single extracted with MeOH
(sample:solvent=1:10 b/v) by UAE for 20 minutes, shorter time required compared
to classical maceration performed in previous research by Sandrasari (2008) in
extracting the antioxidant component from the indigenous vegetables. Velickovic
(2006) stated that the maximum concentration of extractable substances in liquid
extract was reached within 20 minutes of extraction at 40 ± 1 oC. The liquid extracts
of Talinum triangulare (Jacq.) Willd were all dark green in color with different
intensity in each treatments (Figure 2).
Figure 2 (Left-Right) Methanol extract of waterleaf treatment control, PK+RP,
PK+AS, RP+AS, and PK+RP+AS
Analysis of Total Phenolic Compound and Antioxidant Activity
The liquid extracts of antioxidant from five groups were analyzed for its total
phenolic compound based on the Folin-Ciocalteau method previously done by
Shetty et al. (1995). Total phenolic compound was measured by Folin-Ciocalteau
method estimating all flavonoids, anthocyanins, and non-flavonoid phenolic
compounds which present in the extract. Folin Ciocalteau method was based on
oxidation-reduction reaction in which phenolic compounds are oxidized with
simultaneous reduction of phosphotungsten-phosphomolybdate complex in base or
alkaline medium turning the color into blue (Figure 3) that can be measured by
spectrophotometer at 765 nm (Gawron-Gzella et al. 2012). The standard of gallic
acid was used and the result of total phenolic was expressed as milligram equivalent
of gallic acid per gram sample. The absorbance value represented the intensity of
phenolic compound in the sample. Greater amount of phenolic compound gave
higher absorbance value.
The extract of the waterleaf was also tested for its antioxidant activity in
scavenging the radicals by DPPH method. DPPH was a stable free radical dissolves
11
in methanol. DPPH became lighter in color (Figure 3) when an antioxidant
scavenges free radicals by hydrogen donation that can be measured at 517 nm
(Gawron-Gzella 2012). The analysis of reducing power was done by the method of
ferric cyanide reducing power. Antioxidant reduced the complex of ferric cyanide
to Fe2+ ion that could be detected by the formation of Prussian blue color (Figure 3)
measured at 700 nm (Sandrasari 2008). Higher absorbance value indicated greater
reducing power of antioxidant.
Figure 3 (Left-right) Total phenolic assay, DPPH assay, and ferric reducing assay
Total phenolic compound of five different treatments of organic fertilizer
showed variation of the result ranged from 6.00 – 10.76 mg GAE/ g dry sample
(wb) (Table 5). The highest total phenolic was found in PK+RP (10.76 mg GAE/ g
dry sample wb) and the lowest total phenolic was found in PK+AS (6.00 mg GAE/
g dry sample wb). In sequential order the highest to the lowest total phenolic content
was PK+RP, control treatment, PK+RP+AS, RP+AS, and PK+AS. Previous
research conducted by Andarwulan (2010) resulted that the phenolic compound
commonly found in ten indigenous vegetables among them katuk (Sauropus
androgynous (L) Merr), kenikir (Cosmos caudatus H.B.K), kedondong cina
(Polyscias pinnata) was ranged from 0.33 – 0.152 mg GAE/ g fresh weight and the
total phenolic content of waterleaf was 0.489 mg GAE/g fresh weight. Compared
to present research, the total phenolic compound was higher in the present research.
Andarwulan et al. (2010) stated that the phenolic compound in waterleaf was
dominated by kaempferol and very little amount of quercetin. The difference of
total phenolic compound between current and previous research indicated that
different solvent and method of extraction used would result in significant different
amount of both kaempferol and quercetin that could be extracted from the samples.
Ethanol 95 % and classical shaking maceration were used in previous research
while methanol and ultrasound assisted extraction (UAE) were used in current
research. UAE proved to be more effective in extracting the phenolic compound
than classical maceration as previously said by Velickovic et al. (2006) in term of
yielding more extractable component.
The DPPH antioxidant activity was expressed as IC50 value. IC50 value
showed the concentration of antioxidant extract from waterleaf to reduce 50 % of
DPPH radical. Lower concentration indicated stronger antioxidant activity. The
extract that had strongest ability to reduce 50 % of DPPH radical was RP+AS
extract (1170.20 ppm) and the weakest one was PK+AS (2396.54 ppm). The result
in sequential order from the strongest to the weakest based on IC50 value was
RP+AS, control treatment, PK+RP, PK+RP+AS, and PK+AS (Table 5).
12
Table 5 Total phenolic and antioxidant activity
Treatment
Total Phenolic*
IC50 Value**
DPPH
AEAC***
Ferric
Reducing
AEAC**
DPPH
Antioxidant
Activity****
Ferric Reducing
Antioxidant
Activity****
Control
9.75d ± 0.16
1244.12a ± 6.44
16.92c ± 0.12 0.14c ± 0.01
1.74b ± 0.04
0.015c ± 0.000
PK+RP
10.76e ± 0.06
1231.23a ± 12.85
18.65d ± 0.00 0.18e ± 0.01
1.73b ± 0.01
0.018d ± 0.001
PK+AS
6.00a ± 0.13
2349.54b ± 1.17
8.50a ± 0.10
0.01a ± 0.00
1.42a ± 0.02
0.002a ± 0.000
RP+AS
7.36b ± 0.00
1170.20a ± 7.78
15.18b ± 0.09 0.16d ± 0.00
2.06c ± 0.01
0.022e ± 0.000
PK+RP+AS
8.85c ± 0.03
1303.42a ± 265.54
15.24b ± 0.37 0.07b ± 0.00
1.72b ± 0.04
0.008b ± 0.000
PK: cow manure
RP: rock phosphate
AS: husk ash
*mg GAE/g dry sample
**mg/g dry sample
***mg AEAC/g dry sample
****mg AEAC/mg GAE
13
Both DPPH and ferric reducing power antioxidant activity were expressed in
AEAC or Ascorbic acid Equivalent Antioxidant Activity by using vitamin C or
ascorbic acid as the reference standard. The value then calculated in the basis of
one gram of total phenolic to show the antioxidant activity per gram total phenolic
(Table 5). The highest antioxidant activity to scavenge DPPH radical per gram total
phenolic was found in RP+AS group (2.06 ± 0.01 mg AEAC/g GAE). The lowest
was found in PK+AS group (1.42 ± 0.02 mg AEAC/g GAE). Control, PK+RP,
PK+RP+AS treatments followed the order from the highest. While for reducing
power, the group with the strongest reducing power was found in RP+AS (0.022 ±
0.00 mg AEAC/g GAE) followed by PK+RP, control, PK+RP+AS, and PK+AS.
Thus could be inferred that high total phenolic content is not always resulting in
high antioxidant activity, but high ability to scavenge free radical will result in high
reducing power. The highest total phenolic content was found in group PK+RP
(10.76 mg GAE/g dry sample), but its antioxidant was weaker in scavenging DPPH
radical and reducing the ferric (1.73 mg AEAC/g GAE and 0.018 mg AEAC/g
GAE) compared to RP+AS (2.06 mg AEAC/g GAE and 0.022 mg AEAC/g GAE).
Thus could be inferred that the organic fertilizer treatment influenced the secondary
metabolite produced in which the high antioxidant activity resulted from other
components (e.g. vitamin C and carotenoid) rather than only phenolic compounds.
It was found that fertilizer rich in soluble nitrogen (e.g. PK or cow manure) could
decrease the ascorbic acid content since nitrogen supply increased plants’ leaf
density. Carotenoid content was said not being influenced by fertilizer treatments
(Zoran et al. 2014). Compared to other vegetables in previous research, the
antioxidant activity of waterleaf was weaker. Kaempferol existed as the only
dominated flavonoid antioxidant found in waterleaf while beluntas (Pluchea indica
Less.) and kenikir (Cosmos caudatus H.B.K) contained quercetin, miricetin instead
of kaempferol (Andarwulan 2010; Sandrasari 2008). Antioxidant activity of
flavonoid depended on the number and location of phenolic group –OH to
neutralize free radical. Quercetin, miricetin and kaempferol had double bond on C2C3 increasing its ability to scavenge free radical. Structure of 3,4-dihydroxyl
(cathecol in B ring) in those three could act as electron donor for targeted radical
(Amic et al. 2002). The amount of hydroxyl group, however, differed from one to
another (quercetin=5, miricetin=6, kaempferol=4) making kaempferol had less
antioxidant activity compared to quercetin and myricetin resulting that the
antioxidant activity of the waterleaf was weaker among other indigenous vegetables
i.e. beluntas (Pluchea indica Less.) and kenikir (Cosmos caudatus H.B.K).
Table 6 Total flavonoid of the waterleaf in different organic fertilizers (Saleh
2013)
Treatment
Flavonoid (mg QE/g dry weight)
Control
PK + RP
PK + AS
RP + AS
PK + RP + AS
PK: cow manure, RP: rock phosphate, AS: husk ash
10.62a ± 2.8
11.07a ± 3.1
9.10a ± 2.7
8.90a ± 1.2
9.71a ± 1.6
14
Based on ANOVA (Table 5 and appendices), total phenolic value was
significantly different between groups of organic fertilizer (p