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Nithiya
Journal
Pharmacy
Pharmaceutical
SJIF Impact Factor 5.210
Volume 4, Issue 05, 808-822.
Research Article
ISSN 2278 – 4357
EFFECT OF ORGANIC AND INORGANIC FERTILIZER ON
GROWTH, PHENOLIC COMPOUNDS AND ANTIOXIDANT
ACTIVITY OF SOLANUM NIGRUM .L
J.Alphonse Ligoriya Mary and T. Nithiya*
Department of Biochemistry, Dharmapuram Ganambigai Govt Arts College (W),
Mayiladuthurai- 609001. Tamilnadu, India.
ABSTRACT
Article Received on
22 Feb 2015,
The application of organic and inorganic fertilizer to the soil is
Revised on 16 March 2015,
Accepted on 06 April 2015
considered as good agricultural practice because it improves the
fertility of the soil and plant quality. The objective of this study is to
compare the effect of organic (vegetable waste, cattle dung) and
*Correspondence for
inorganic fertilizer (NPK) on Solanum nigrum. The experiment was
Author
T. Nithiya
arranged in a randomized design in three replicates. Parameters assess
Dept. of Biochemitry,
include leaf area, height, leaf length, nutrient composition,
Dharmapuram
phytochemicals and antioxidant activity on leaf of Solanum nigrum.
Ganambigai Govt. Arts
The application of cattle dung and vegetable waste gave plants with
College (w),
Mayiladuthurai- 609001.
the greatest plant height, leaf area and surface length. The mean values
recorded for these parameters were 38±3.16, 10.5±2.18cm &
Tamilnadu, India.
5.5±1.58cm for organic fertilizer and 36±3.14 cm 8.0±1.12cm &
4.0±1.48cm for inorganic fertilizer. Organic manuare produced higher effect on carbohydrate,
protein, amino acid, vitamin c and chlorophyll content in the leaf of Solanum nigrum when
compared with NPK fertilizer. Mean values recorded for the above parameters in leaf sample
were 30.0±2.41, 1.93±1.93, 3.31±0.65, 2.53±0.72, 1.24±0.53mg/100g compared with values
of 10.0±2.26, 1.25±1.62, 2.0±0.52, 2.26±0.80, 1.98±0.40mg/100g respectively for inorganic
fertilizer.
Effect of organic fertilizer in plant phytochemicals was higher than that of
inorganic fertilizer. Values obtained for alkaloids, flavonoids, tannins, saponins and total
phenol in organic treated leaf samples were 0.21±0.80, 0.9±0.53, 0.52±0.16, 0.34±0.36,
0.26±0.18mg/100g, respectively as against values of 0.2±0.32, 0.5±0.46, 0.4±0.12,
0.29±0.26, 0.8±0.14mg/100g for inorganic fertilizer. Organic fertilizer treated plants have
higher antioxidant activity than the inorganic fertilizer. Mean values recorded for leaf sample
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in organic fertilizer were 64.21±15.81, 35.42±12.80mg/100g respectively corresponding
mean values for inorganic leaf sample were 42.12±14.72, 25.42±7.90mg/100g.
The
experimental results of this study have showed that organic fertilizer produced higher effect
on Solanum nigrum leaf when compared with inorganic fertilizer. High amounts of
phytochemicals, nutrients and antioxidants recorded in this study gives preference to the use
of organic than inorganic fertilizer.
KEYWORDS: Solanum nigrum, Nutritional status, phytochemicals & In vitro antioxidant
activity. Organic and inorganic fertilizer.
INTRODUCTION
Declining soil fertility resulting from continuous cultivation of small holder farms and
escalating cost of imported fertilizers and the need to conserve and build natural resource
capital and biodiversity, has led to renewed interest in the use of local nutrient resources for
soil fertilizer management.[1] The use of organic matter such as animal manures, human
waste, food wastes, yard wastes, sewage sludges and composts has long been recognized in
agriculture as beneficial for plant growth and yield and the maintenance of soil fertility. The
new approaches to the use of organic amendments in farming have proven to be effective
means of improving soil structure, enhancing soil fertility and increasing crop yields. Organic
matter are excellent source of plant-available nutrients and their addition to soil could
maintain high microbial populations. Crop yields have increased with the corresponding
improvements in soil quality from additions of organic matter. Significant yield increases
using mulches from coffee husks and increases in productivity using animal manures and hay
residues have been reported. Their important roles in the soil and their potentially positive
effect on crop yields have made organic amendments a valuable component of farm
fertilization and management programs in alternative agriculture.[2]
The increasing global interest and expanding market of herbal drugs have led to their
introduction into cultivation to meet the demand at reasonable economic price. Cultivation of
medicinal plants can also facilitate in maintaining standard-in quality, potency and chemical
composition of the produce. While are in constant endeavour to increase production, seldom
the issue of quality of crops is studied. In medicinal plants, the quality of end product is very
important; the secondary metabolites from medicinal and aromatic plants are valued in
commerce. Biofertiliser, domestic and industrial waste application are commonly employed
upgradation technique to medicinal and aromatic plants.[3]
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Solanum nigrum is an important leafy vegetable. It has been traditionally used as analgesic,
antispasmodic, antiseptic, antidysentric, emollient, diuretic, soporific, laxative, anticancer,
anti-ulcer and disorders of neuro-vegetative system.[4] This medicinal value is attributed to
the alkaloidal contents of the plant. In India Solanum nigrum mixed with other herbal
medicine has shown to be hepatoprotective in cirrhotic patients. This protective effect can be
attributed to the diuretic, antiflammatory, anti-oxidative and immune modulating properties
of the component herbs.[5] The fruits extracts of Solanum nigrum have anti-tumor and
neuropharmacological properties and can be used as an anti-oxidant and cancer chemoprevention matter.[6] Hence, the present study was taken to investigate the effect of organic
and inorganic fertiliser on growth, secondary metabolites and antioxidant status of Solanum
nigrum L.
MATERIAL AND METHODS
The experiment was carried out at the D.G.G.Arts College (W) in Mayiladuthurai, The study
was carried out between November 2014 to February 2015 in the green house in experimental
pots. The experiment was conducted using 5-litre pots containing 5kg of soil. Cattle dung and
vegetable waste compost were applied at the rates of 1.5kg per 5kg of soil one week before
sowing, while NPK fertilizer was applied at the rate of 3g per 5kg of soil three weeks after
sowing. Ten viable seeds of Solanum were planted in each of the pots. Harvesting was done
until the 8th week of plant age. The following parameters were observed in leaf of organic
and inorganic treated Solanum nigrum plant.
Nutritional Analysis
Estimation of carbohydrate
1g of dried and powdered sample was extracted with 10ml of distilled water in water bath for
15min. The supernatant was used as the test sample. 1ml of the supernatant was taken and
4ml of anthrone reagent was added to it then boiled for 10min and then cooled at room
temperature. Absorbance was measured at 620nm wavelengths. Carbohydrate was reported as
mg/g of dry weight.[7]
Estimation of protein
1.0gram of leaf powder was ground using pestle and mortar with 1.0 ml of phosphate buffer.
These samples were kept overnight for complete extraction of protein. These were
centrifuged for 20 minutes. The supernatant is used for protein analysis and the pellet is
discarded.[9] To 1.0 ml of supernatant from above is added 5.0 ml of alkaline copper sulfate
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reagent and thoroughly mixed. Allowed to stand for ten minutes and then add 0.5 ml of
Folin.s reagent. In order to develop colour this is kept standing for 30 minutes. This was
followed by recording absorbance in colorimeter at 660 nm, against a blank. The blank is
prepared by taking 1.0mL of 0.5 M NaOH in place of sample in cuvette. Bovine serum
albumin is used to draw a standard curve and the amount of proteins in samples are
estimated.
Estimation of amino acids
500mg of plant materials were weighed and macerated with a pestle and mortar with 10ml of
80% ethanol. The homogenate was centrifuged for 10minutes at 800g. The supernatant was
saved and the extract was used for the estimation of amino acids. 1ml of the extract was
pipetted out into a test tube. A drop of methyl red indicator was added. The sample was
neutralized with 1ml of 0.1N sodium hydroxide. To this, 1ml of ninhydrin reagent was added
and mixed thoroughly. The content of the test tube was heated for 20minutes in a boiling
water bath. 5ml of the diluents solution was added and heated in water bath for 10minutes.
The test tubes were cooled and the contents were mixed thoroughly. Blank was prepared with
1ml of distilled water (or) ethanol. The absorbance was read at 570nm in a colorimeter.[10]
Determination of ascorbic acid
The ascorbic acid content was measured using a modified method of.[11] The fresh leaf
samples (1g) were extracted in 1% of phosphate-citrate buffer, pH 3.5 using a pestle and
mortar. The homogenate was filtered. The filtrate was added to 1.7mM 2,6
dichloroindophenol (2,6-DCPIP, 1mL) in a 3ml cuvette. The absorbance at 520nm was read
within 10min of mixing the reagents. The extraction buffer was used as a blank. L Ascorbic
acid was used as a standard. Ascorbic acid was recorded as mg/g L-ascorbic acid in fresh
leaves.
Estimation of chlorophyll
0.5gm fresh plant material was homogenized in 20ml of 80% chilled acetone with the help of
mortar and pestle in dark. A pinch of MgCO3 powder added. The extract filtered through
Whatman no.1 filter paper. Final volume of the filtrate was made to 100ml with 80% acetone.
Absorbance reading done at the 645nm wavelength respectively with 80% acid used as a
blank.[12]
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Phytochemical analysis
Determination of total phenolics
The concentration of total phenolics was examined spectrophotometrically employing the
Folin–Ciocalteu reagent protocol as reported by.[13] In this assay, dry mass of leaves extract
(50mg) was mingled first with 0.5ml of Folin-Ciocalteu reagent and then with 7.5ml of
deionized water. The whole mixture was placed at room temperature for about 10min. After
this, 1.5ml of 20% sodium carbonate was also added. The mixture was then heated in a water
bath at 40°C for 20min and then it was cooled in an ice bath. At the end, the absorbance was
read at 755nm with a spectrophotometer. Amount of TP was calculated by means of
designing a calibration curve of gallic acid. The TPC results were shown with reference to
gallic acid equivalents (GAE) per dry matter.
Determination of total flavanoids
Total flavonoid was conducted according to.[14] using aluminium chloride colorimetric
method. 1ml of extracts was added with 4ml of distilled water in a flask. Then, 0.3ml of 5%
NaNO2 was added. After 5min, 0.3ml of 10% AlCl3 was added and after 6min, 2ml of 1M
NaOH was added. The mixture was diluted to 10ml with distilled water. The absorbance of
the solution was measured at 510nm using a spectrophotometer. The results were expressed
as mg catechin equivalents (CE)/g samples.
Determination of total tannins
Tannin content of the given sample was estimated by following the standard procedure.[15]
The sample extract (1 ml) was mixed with Folin-Ciocalteau’s reagent (0.5 ml), followed by
the addition of saturated Na2CO3 solution (1 ml) and distilled water (8 ml). The reaction
mixture was allowed to stand for 30 min at room temperature. The supernatant was obtained
by centrifugation and absorbance was recorded at 725 nm using UV-Visible
Spectrophotometer. Increasing concentrations of standard tannic acid was prepared and the
absorbance of various tannic acid concentrations was plotted for a standard graph. The tannin
content was expressed as mg tannic acid equivalent per 100 gram of the sample.
Determination of total alkaloid content
The total alkaloid content was determined according to UV Spectrophotometer method.[16]
This method is based on the reaction between alkaloid and bromocresol green. The part of
the plant extract was dissolved in 2N HCl and then filtered. 1ml of this solution was
transferred to separatory funnel and washed with 10ml chloroform The pH of phosphate
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buffer solution was adjusted to neutral with 0.1N NaOH. One ml of this solution was
transferred to a separating funnel and then 5ml of bromocresol solution along with 5ml of
phosphate buffer were added. The mixture was shaken and the complex formed was
fractioned with chloroform by vigorous shaking. The fractions were collected in a 10ml
volumetric flask and diluted to volume with chloroform. The absorbance of the complex in
chloroform was measured at 470nm.The results were averaged and reported in the form of
mean ± S.E.M.
Determination of saponin
Saponin determine by the method of.[17] 20g of plant powder was put into a conical flask and
100ml of 20% aqueous ethanol were added. The samples were heated over a hot water bath
for 4h with continuous stirring at about 550C. The mixture was filtered and the residue reextracted with another 200ml 20% ethanol. The combined extracts were reduced to 40ml over
water bath at about 900C. The concentrate was transferred into a 250ml separatory funnel and
20ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered
while the ether layer was discarded. The purification process was repeated. 60ml of n-butanol
was added. The combined n-butanol extracts were washed twice with 10ml of 5% aqueous
sodium chloride. The remaining solution was heated in a water bath. After evaporation the
samples were dried in the oven to a constant.
In vitro antioxidant assay
DPPH radical scavenging assay
The free radical scavenging activity was measured by using the method of.[18] Briefly, 0.1mM
solution of DPPH• in ethanol was prepared and 1ml of this solution was added to 3ml of leaf
extract. The mixture was shaken vigorously and allowed to stand at room temperature for 30
min. Then the absorbance was measured at 517nm in a spectrophotometer. Lower absorbance
of the reaction mixture indicated higher free radical scavenging activity. The DPPH free
radical scavenging activity was calculated using the following formula
% scavenging = [Absorbance of control - Absorbance of test
sample/Absorbance of control] X 100
Reducing power assay
The reducing power of leaf of Solanum nigrum was determined according to the method of[19]
1ml of methanol leaf extract was dissolved in 1ml of distilled water, phosphate buffer (2.5
ml,0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mixture was
incubated at 50 ◦C for 20 min. A portion (2.5 ml) of TCA (10%) was added to the mixture,
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which was then centrifuged for 10 min at 1000 × g .The upper layer of solution (2.5 ml) was
mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and the absorbance was
measured at 700 nm in a spectrophotometer. Higher absorbance of the reaction mixture
indicated greater reducing power.
RESULTS AND DISCUSSION
Height, Leaf area and Leaf length
Table-1 show the effect of cattle dung, vegetable waste compost and NPK fertilizer on the
performance of Solanum nigrum. At week 6 after sowing, the application of vegetable waste
and cattle dung produced the greatest plant height (38±3.16 cm) while the lowest plant height
was obtained from the NPK treatment (36±3.14 cm) (Fig-1).
This results is in agreements with previous reports,[20] reported that plant height of Okra was
greater in poultry dropping treated soil. They attributed it to the ready availability of nutrients
for the easy absorption by plant root, thus resulting in an increase in plant growth.[21] Asserted
that organic manuare treated soil could increase plant height when compared with other
sources of fertilizer. Comparatively high nitrogen content of poultry manure buttresses the
vegetative growth of crops.[22] Reported a positive effect of organic fertilizer on vegetative
growth. The plants treated with NPK fertilizer grew fastest in the first three weeks. The plants
treated with organic fertilizer grew higher from the sixth week up to the end of the
experiment in week 10.
Table -1 showed the effect of organic (vegetable waste and cattle dung) and inorganic (NPK)
fertilizer on leaf area and leaf length. The greatest leaf area and leaf length was observed in
the organic fertilizer treatment (10.5±2.18cm & 5.5±1.58cm) and lowest in the inorganic
fertilizer treatment (8.0±1.12cm & 4.0±1.48cm). Inceased leaf area and leaf length implies
higher light interception and dry matter product which invariably promotes plant growth.[23]
Table 1: Effect of organic and inorganic fertilizer on morphological traits of Solanum
nigrum
S.No
1
2
3
Parameters
Plant height (cm)
Leaf length (cm)
Surface area (cm)
Organic fertilizer
38±3.16
10.5±2.18
5.5±1.58
Inorganic fertilizer
36±3.14
8 ±1.12
4±1.48
Values are expressed as mean ± standard deviation
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Fig 1 : Effect of organic and inorganic fertilizer morphological traits of Solanum nigrum
The response of Solanum nigrum to organic manuare application may also be attributed to the
increasing total organic matter, macro and micronutrients rendered after the application of
manure.
Nutritional status, Phytochemicals & In vitro antioxidant activity
Results in Table- 2 indicate the effect of cattle dung, vegetable waste manuare and NPK
fertilizer on nutritional content of Solanum nigrum. Organic manuare produced higher effect
on carbohydrate, protein, amino acid, chlorophyll, vitamin c and chlorophyll content in the
leaf of Solanum nigrum when compared with NPK fertilizer. Mean values recoreded for the
above parameters in leaf sample were 30.0±2.41, 1.93±1.93, 3.31±0.65, 2.53±0.72,
1.24±0.53mg/100g respectively compared with values of 10.0±2.26, 1.25±1.62, 2.0±0.52,
2.26±0.80, 1.98±0.40mg/100g respectively for inorganic fertilizer (Fig-2).
The result of comparative effect of organic and inorganic fertilizer on phytochemicals of
Solanum nigrum was given in Table- 3. Effect of organic fertilizer in plant phytochemical
was higher than that of inorganic fertilizer.
Values obtained for alkaloids, flavonoids,
tannins, saponins and total phenol in organic treated leaf samples were 0.21±0.80, 0.9±0.53,
0.52±0.16, 0.34±0.36, 0.26±0.18mg/100g, respectively as against values of 0.2±0.32,
0.5±0.46, 0.4±0.12, 0.29±0.26, 0.8±0.14mg/100g for inorganic fertilizer.
Effect of organic and inorganic fertilizer on antioxidants of Solanum nigrum was shown in
Table-4. Results revealed a similar trend of higher mean values for organic fertilizer when
compared with inorganic fertilizer. Mean values recorded for leaf sample in organic fertilizer
were 64.21±15.81, 35.42±12.80mg/100g respectively. Corresponding mean values for
inorganic leaf sample were 42.12±14.72, 25.42±7.90mg/100g.
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Preceding results are in harmony with those obtained by[24] on African marigold (Tagetes
erecta) who cited that, compost treatment increased chlorophyll a and b, carotenoids content,
total carbohydrate. Also,[25] on rosemary plants,[26] on Montana plants found that, compost
treatment increased plant pigments and total carbohydrate over NPK treatment plants and[27]
on tomato and cucumber found that, application of compost resulted in an increment over
control plants. Hereabout[28] on Barley and Maize and[29] on Tobacco mentioned that,
application of organic manuare increased photochemical activity of chloroplasts, total
chlorophyll content as well as total carbohydrate.
This study on comparative effect of organic and inorganic fertilizers on nutritional analysis,
phytochemicals, in vitro antioxidant level of Solanum nigrum revealed that organic fertilizer
produced higher effects on all the parameters investigated when compared with inorganic
fertilizer. The results of this study are in agreement with[30-37] who have reported increases
with organic fertilizers on phytochemical, nutritional and in vitro antioxidant. These increases
could be due to the ease with which nutrients such as N, P and K in NPK fertilizers are lost
by leaching. Nutrients in organic material are less easily available since the materials have to
be decomposed and organic nutrients mineralized.[38] Organic manures activate many species
of living organisms which release phytohormones and may stimulate the plant growth and
nutrients[39] and such organisms need nitrogen for multiplication[40] The positive correlation
shown in Table-3 between total phenolic and flavonoid compounds indicates that an increase
in phenolic was followed by an increase in total flavonoid. Both were found to be highly
correlated with antioxidant activity. Among Medan and Sri Pontian, it was found that Medan
had higher total phenolics and total flavonoids. Phenolic compounds and flavonoids in this
condition may play an important role as scavengers for free radicals and other oxidative
species.[41] Phenolic molecule is characteristic of a plant species or even of a particular organ
or tissue of the plant.[42]
Results of this study are also in consonance with results of the biggest and most extensive
scientific study and research into the benefits of organic food by,[43] who reported that
organic food is more nutritious than non organic (ordinary produce) food and may in fact
lengthen peoples lives. She also found that they contain higher levels of antioxidants and
flavonoids which help ward off heart disease and cancer as well as iron and zinc. Research
that was carried out in the Newcastle University also showed that organic food contain more
antioxidants and less unhealthy fatty acid[44] This could be attributed to the fact that the
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nutrients in the organic manure are released gradually through the process of mineralization
maintaining optimal soil levels over prolonged periods of time. Some of the organic
substances released during the mineralization may act as chelates that help in the absorption
of iron and other micro-nutrients.
Table 2: Effect of organic and inorganic fertilizer on nutritional profile of Solanum
nigrum
S. No
1
2
3
4
5
Parameter
Carbohydrate
Protein
Amino acid
Chlorophyll
Ascorbic acid
Organic Fertilizer
30.0±2.41
1.93±1.93
3.31±0.65
2.53±0.72
1.24±0.53
Inorgani Fertilizer
10.0±2.26
1.25±1.62
2.0±0.52
2.26±0.80
1.98±0.40
Values are expressed as mean ± standard deviation
Fig 2: Effect of organic and inorganic fertilizer on nutritional profile of solanum nigrum
Table 3: Effect of organic and inorganic fertilizer on phytochemicals of Solanum nigrum
S.No
1
2
3
4
5
Parameter
Alkaloids
Flavonoids
Tannins
Saponins
Total phenol
Organic Fertilizer
0.21±0.80
0.9±0.53
0.52±0.16
0.34±0.36
0.26±0.18
Inorganic Fertilizer
0.2±0.32
0.5±0.46
0.4±0.12
0.29±0.26
0.8±0.14
Values are expressed as mean ± standard deviation
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Fig 3: Effect of organic and inorganic fertilizer on Phytochemical of
Solanum nigrum
Table: 4. Effect of organic and inorganic fertilizer on in vitro antioxidant of Solanum
nigrum
S.No Samples
DPPH
1
Organic fertilizer
64.21±15.81
2
Inorganic fertilizer
35.42±12.80
Values are expressed as mean ± standard deviation
Reducing power assay
42.12±14.72
25.42±7.90
Fig 4: Effect of organic and inorganic fertilizer on in vitro antioxidant of Solanum
nirgum
CONCLUSION
Fertilisation is the most important and controllable factor affecting nutritional value of plants.
The result indicate that application of cattle dung manure and vegetable waste compost can
enhance the growth parameter, nutritional content, phytochemicals and antioxidant activity of
Solanum nigrum. The effects of composts on plants are not solely attributed to the quality of
mineral nutrition is provided but also to its other growth regulating components. Furthermore,
the application of compost in the field enhances the quality of soils by increasing microbial
activity and microbial biomass which are key components in nutrients cyclings, production of
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plant growth regulators. Therefore, it could be concluded that, the chemical fertilizers of NPK
could be replaced by the compost for improving the quality of the produced yield under safe
agriculture conditions, in addition to decreasing the production costs and environmental
pollution.
ACKNOWLEDGEMENT
We take this opportunity our sincere thanks and gratitude to Dr. A.Malarvizhi, Head and
Asst professor, Department of Biochemistry, Dharmapuram Ganambigai Govt Arts College
(W), for valuable guidance and support for my research work.
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