Author’s Accepted Manuscript

Humic acid and vermicompost increased bioactive

components, antioxidant activity and herb yield of
Chicory (Cichorium intybus L.)

Hossein Gholami, Mohammad Jamal Saharkhiz,

Fatemeh Raouf Fard, Askar Ghani, Fatemeh Nadaf
www.elsevier.com/locate/bab

PII: S1878-8181(17)30574-1
DOI: https://doi.org/10.1016/j.bcab.2018.03.021
Reference: BCAB733
To appear in: Biocatalysis and Agricultural Biotechnology
Received date: 7 November 2017
Revised date: 22 March 2018
Accepted date: 26 March 2018
Cite this article as: Hossein Gholami, Mohammad Jamal Saharkhiz, Fatemeh
Raouf Fard, Askar Ghani and Fatemeh Nadaf, Humic acid and vermicompost
increased bioactive components, antioxidant activity and herb yield of Chicory
(Cichorium intybus L.), Biocatalysis and Agricultural Biotechnology,
https://doi.org/10.1016/j.bcab.2018.03.021
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 Humic acid and vermicompost increased bioactive components,

antioxidant activity and herb yield of Chicory (Cichorium intybus L.)

Hossein Gholami1, Mohammad Jamal Saharkhiz1,2*, Fatemeh Raouf Fard1, Askar

Ghani3, Fatemeh Nadaf1

1
Department of Horticultural Sciences, Faculty of Agriculture, Shiraz University, Iran
2
Medicinal Plants Processing Research Center, Shiraz University of Medical Sciences,

Shiraz, Iran

3
Department of Horticultural Science, College of Agriculture, Jahrom University,

Jahrom, Iran

*
Corresponding author. M.J. Saharkhiz, Ph.D., Professor of Medicinal and Aromatic Plants

Production and Processing, Department of Horticulture, Faculty of Agriculture, Shiraz University,

Postal Code: 71441-65186 Shiraz, Iran. Tel.: +98 713 613 8144; fax: +98 713 228 6133.

saharkhiz@shirazu.ac.ir

Abstract

Chicory (Cichorium intybus L.) is one of the most important plants in the

medicinal, nutritional and cosmetic industry. This study was conducted to evaluate

the effects of humic acid (HA) and vermicompost on the herb yield and phenolic

components of chicory aerial parts. Plants were grown under 4 humic acid

treatments (0, 0.3, 0.6 and 0.9 kg/ hectare) and 4 vermicompost levels (0, 5, 7.5 and

10 ton/ hectare) with three replications. Measurement of traits included fresh and

dry yields of aerial parts, the IC50 and inhibition percentage of free radicals, the

1
total and individual amounts of phenolics and flavonoids (caffeic, gallic, ellagic

and p- coumaric acid, catechin, flavones and flavonols). Results show that humic

acid and vermicompost improve the chicory yield and phytochemical properties

such as the total contents of phenolics and flavonoids. The highest yield of dry

aerial parts (20.29gr per plant) was achieved by 0.9 kg per hectare humic acid in

combination with 10 tons per hectare vermicompost. The highest total contents of

phenolics and flavonoids were achieved when humic acid was applied at 0.9

kg/hectare in addition to vermicompost at 7.5 ton/ hectare. The content of caffeic

acid was highest among other phenolic components. The highest caffeic acid was

achieved by treatments of 0.3 kg per hectare humic acid combined with 5 tons per

hectare vermicompost. The content of ellagic acid in shoots was highest when

vermicompost was applied alone, by 5 tons per hectare, with an average yield of

262.51 mg/100 g DW.

Keywords: Humic acid, Vermicompost, Flavonoids, Chicory, Antioxidant

activity

1- Introduction

The interest in medicinal plants for pharmaceutical production is growing in

most countries all over the world. Over the past one hundred years, a great number

of researchers have studied medicinal plants and the pharmaceutical drugs that can

be derived from them. Medicinal plants with natural origin open new horizons for

the medical and pharmaceutical community. According to the World Health

Organization (WHO, 1998), one quarter of the prescribed drugs in industrialized

2
countries have compounds originating directly or indirectly (semi-synthesized)

from plants. During the past few decades, the overuse of chemical components

such as fertilizers has resulted in the spoiling of water resources, along with soil

and its bio sources, and the decrease in qualities of food products. Irreversible

damages to ecosystems are another problem in addition to the mentioned issues

(Sharma, 2002). Therefore, sustainable agriculture and alternative methods based

on the use of organic fertilizers could be considered as the optimal solution to

overcome this problem in order to eliminate or significantly reduce the use of

chemical inputs. Common knowledge states that sustainable agriculture is

encountering a challenge regarding the increase in the quality of products and yield

stability. Medicinal plants are beneficial options for this system of sustainable

agriculture (Gupta et al., 2002). Many researchers believe that the plants cultivated

in organic agricultural systems often produce more secondary metabolites than

plants cultivated in conventional farming systems (Adam, 2001).

Humic acid is known as a natural compound which is the result of decay of

organic matter in soil, peat and lignin. It could be used in sustainable agriculture

(Nardi et al., 2002). Before acting as a fertilizer, humic acid acts as a soil

amendment which helps to increase the number of microorganisms in the soil,

thereby improving the physical conditions of the soil. It can also adjust soil pH,

and would affect the activity and/or the concentration of enzymes and hormones. It

regulates plant growth, and increases water use efficiency. These would

consequently increase plant resistance to drought and salinity stress, which lead to

the increase in nutrient absorption, and the increase in germination and root

growth. All of these attributes would ultimately improve the quantity and quality of

products (Cangi et al., 2006; Zhang et al., 2010 and Eneji et al., 2013).

3
 Vermicompost is a kind of compost produced by a red roundworm called

Eisenia foetida (Garg et al., 2006). Vermicompost has high porosity and high water

holding capacity. It has a good ability to absorb and maintain nutrients, with proper

capacities for ventilation and drainage. Vermicompost increases the availability of

nitrogen and phosphorus for plants by nitrogen fixation, and it dissolves

phosphorus while giving off no unpleasant odor or stink in the process. It is free

from any pathogen, and is suitable to be used in sustainable agriculture because of

its ability to enhance soil quality and nutritional conditions, which can result in

higher growth and yield for medicinal plants and crops (Akbarinia et al., 2004;

Mcginnis et al., 2003 and Prabha et al., 2007). There are some reports about the

presence of vitamin B12 and growth regulators in vermicompost (Tomati et al.,

1988 and Atiyeh et al., 2000). Physicochemical properties of humic substances in

vermicompost improves the plants’ yield, probably by increasing the storage

capacity of nutrients and growth regulators (Tomati et al., 1983 and Arancon et al.,

2005) in line with an increase in the dynamics of soil microorganisms (Arancon et

al., 2004). In addition, vermicompost is known to exert buffering properties that

are able to prevent changes in soil pH and can increase the absorption of alkali

cations in the soil sediment (Bouwman & Reinecke, 1991).

Chicory, with the scientific name of Cichorium intybus L., is a plant from the

Asteraceae family (Ghahraman, 1994). The Cichorium genus includes six species.

Among them, however, C. intybus is the most popular. Species that have already

been studied by other researchers are C. intybus L., C. endivia L. and C. pumilum

Jacq. (Kisiel et al., 2004). Chicory can be annual, biennial or perennial, depending

on its life conditions. It is a cool season plant that grows in humid regions with low

altitude (Finke et al., 2002). So far, more than 100 pharmaceutical compounds have

4
been identified and extracted from the chicory. The most important of these

compounds are polysaccharides such as inulin, chicoric acid, sesquiterpene

lactones, coumarins, flavonoids, and vitamins (Malarz et al., 2002 and Velayutham

et al., 2006). Pharmacological studies conducted on the chicory showed that this

plant has medicinal features including antioxidant activity (Cavin, 2005 and

Heimler et al., 2009), besides having properties that are anti-cancerous (Ahmed,

2009), antimicrobial (Kim et al., 1999), antifungal (Nishimura & Satoh, 2006),

anti-inflammatory (Hassan, 2008), anti-malarial (Bischoff et al., 2004), anti-

allergic (Gazzani et al., 2000), and antidiabetic (Petrovic et al., 2004 and Pushparaj

et al., 2007). It can also be used in the treatment of AIDS (Malarz et al., 2002 and

Velayutham et al., 2006), and in preventing the proliferation of the Herpes Simplex

Virus (HSV) (Ziai et al., 2007). Chicory is known to be neuroprotective (Marteau

et al., 2011), and has anti-testicular toxicity (Chopra et al., 1956) and anti-

hepatotoxicity (Ahmed et al., 2003). It is a diuretic (Kaur & Gupta, 2002), laxative

(Sugatani et al., 2004), and protects liver cells from oxidative damage (Zafar & Ali,

1998).

Nowadays, production of medicinal and aromatic plants by using organic

fertilizers is a very important and essential topic to obtain medicinal crops with

high quality and free of chemical residues. Therefore, the aim of the present work

was to determine the herb yield and variation in concentration of valuable

secondary metabolites of Cichorium intybus by using humic acid and

vermicompost fertilizers under field conditions.

2- Materials and methods

5
 2.1. Plant material

To study the effects of vermicompost and humic acid application on

biochemical characteristics of chicory, a factorial experiment was set up in a

randomized complete block design (RCBD) in the field of Horticulture Research

Station at Shiraz University located in Badjgah with geographical characteristics of

38° 29' northern latitude and 35° 52' east longitude. Altitude measured 1810 meters

above mean sea level. According to the fertilizer requirement of plants (Table 3),

along with the obtained data of soil (Table 1) and analysis of vermicompost

properties (Table 2), four levels of vermicompost were applied, including control

(without fertilizer), 5, 7.5 and 10 tons per hectare. Also, four levels of humic acid

fertilizer (HUMAX 95-WSG, Lot No: 410143, JH Biotech, Inc ® Ventura,

California, USA) were considered according to the manufacturer’s instruction,

including the control, 0.3, 0.6 and 0.9 kg per hectare, in three replications, adding

up to a total of 48 experimental units. Seeds of C. intybus were collected from an

herbal garden of Jahrom University. The seeds were sown in April, 2016. The plots

size was 2.1 m2 (3.5m×0.6m) with a row to row and plant to plant distance of 1.5 m

and 30 cm, respectively. Vermicompost treatments were applied to a depth of 15

cm in the soil. The plants were irrigated twice a week until the flowering was

observed, and thereafter irrigation was performed three times a week until

flowering time, depending on weather conditions. The aerial parts of the plants

were harvested at flowering stage on July, 2016. The plant species was identified

and authenticated by A. Khosravi, a plant taxonomist at Shiraz University, Shiraz,

Iran. The voucher specimen was deposited in the herbarium.

2.2. Determination of IC50, inhibition percentage of free radicals (% I), total

phenols, flavones, flavonols, and total flavonoids

6
 In order to prepare the extract of plant samples for measuring the biochemical

factors, methanol 70% was used at a ratio of 5:1 (volume- weight) as solvent

(Wojdyło et al., 2007). Determination of free radical scavenging was performed by

using the DPPH test according to Oke et al (2009). DPPH was used by the

concentration of 5mM. Accurate amount of DPPH should be solved in methanol

70% (v/v), then mixed 20 microliter extract with 180 microliter DPPH solution and

leave for 30 min in dark place. Methanol 70% was used as control. Samples’

absorptions were read at a wavelength of 517 nm with Epoch Microplate

Spectrophotometer, BioTek Instruments, Inc., USA. The inhibition percentage of

the free radicals (I%) was calculated by using the following formula:

Inhibition percent of free radicals = [(absorption number of samples -

absorption number of control) / absorption number of control] ×100

Concentration that can neutralize free radicals by 50% (IC50 is a parameter for

comparing the antioxidant activity of the extracts) was calculated and reported by

using the graphical and linear equation based on mg of dry samples in a 70 ml of

methanol. Finally, the value of IC50 obtained from extracts were compared to the

IC50 of BHT synthetic antioxidants as a positive control (Hashemi et al., 2011).

Measuring the total phenols was performed according to the Folin’s reagent

method and the use of Gallic acid as standard (purchased from the brand MERCK,

Germany) by using a spectrophotometer at the wavelength of 765 nm (Wojdyło et

al., 2007).

The method proposed by Menichini et al (2009) was used for measuring the

total flavonoid by means of a spectrophotometer at a wavelength of 510 nm

through a standard curve of quercetin from Sigma-Aldrich. The amounts of

7
flavones and flavonols were measured (by standard curve of quercetin which

purchased from Sigma-Aldrich) based on Popova et al (2004) at the wavelength of

425 nm. The reaction mixture (2.5 mL), 1 mL methanolic extract and 1 mL

aluminums chloride (2%) in methanol (w/v) were mixed and the volume was made

up to 2.5 mL with methanol.

2.3. Extraction and determination of phenolic components

Extraction was done by using a solvent (85% methanol + 15% acetic acid) with

a concentration that was 10 times more than the samples. In the next step, the

micro tubes containing extract were placed in ultrasonic bath (Bandelin

manufacturer, Germany) for 15 min at low temperature and in the dark so as to

isolate phenolic compounds from plant tissue, and then to solve them in the

solvent. The samples were kept at a temperature of 0 °C for 20 minutes and were

centrifuged at 10,000 rpm. The supernatant was removed and the n-hexane phase

(same as the upper phase) was added. Again, the samples were centrifuged at

10,000 rpm for 10 minutes at 0 °C. At this stage, a two-phase solution was formed:

the upper phase mixture was hexane and the lower phase was polyphenols. Then,

polyphenols were removed with the help of a syringe which had a filter on its head.

The polyphenols were then transferred to glass containers and were injected into

the chamber of an HPLC system device (Justesen et al., 1998). The standard for all

8
five phenolic acids (caffeic acid, p- coumaric acid, ellagic acid, catechins, and

gallic acid) were used based on the Sigma-Aldrich brand.

2.3.1. Identification of phenolic components using high performance liquid

chromatography (HPLC)

To isolate and measure the amounts of polyphenols by the HPLC device

(model Smartline), the pump 1000 manufactured by Knauer, German was used.

The column Eurospher was 100-5 C18, with a length of 250 mm and an inner

diameter of 4.5 mm, and a diameter of 5 micrometers. The UV detector was set at a

wavelength of 280 nm with an oven temperature of 30 ° C. Accordingly, a gradient

elution program was selected with the elution rate of 1 ml per min and an injection

volume of 20 ml. The mobile phase gradient of methanol and formic acid was 1%.

A comparison of sample chromatogram peak retention times was performed with

the standards (qualitative analysis), and the area under the curve for each sample

placement in the standard curve contributed to quantitative analysis. Finally, the

concentrations of phenols as milligrams per hundred grams of dry weight (mg/100

g DW) were calculated and reported.

2.4. Statistical analysis

Data were presented as mean values ± standard error for three replicates. The

significance of differences was determined by Duncan’s Multiple Range test by

using a general linear model (GLM) procedure and a 0.05 level of significance (p <

0.05) in SAS, version 9.4 for Windows. Correlation coefficients were estimated for

9
each pair of traits as the Pearson product moment correlation coefficient by using

SAS.

3- Result and discussion

Results of the interactions between fertilizers and their effects on fresh and dry

aerial parts showed that the highest fresh yield of aerial parts occurred by

treatments of 0.9 kg per hectare humic acid which interacted with 10 tons per

hectare of vermicompost, and 0.6 kg per hectare humic acid interacted with 7.5

tons per hectare vermicompost, thereby yielding averages of 200.77 and 195.74 gr

per plant, respectively, which showed no significant difference compared to one

another (Table 5). The highest yields of dry aerial parts were averages of 20.29,

18.35 and 17.98 gr per plant, which were achieved by treatments of 0.9 kg per

hectare humic acid in combination with 10 tons per hectare vermicompost, 0.6 kg

per hectare humic acid in combination with 7.5 tons per hectare vermicompost, and

0.9 kg per hectare humic acid in combination with 7.5 tons per hectare

vermicompost, respectively (Table 5). These also showed no significant difference

compared with one another

Furthermore, the interaction between the two factors, i.e. vermicompost and

humic acid, in this experiment indicated that the inhibition percentage of free

radicals and also the content of IC50 in shoots suggested that the combined

application of vermicompost and humic acid is highly effective on increasing the

antioxidant power of the plant (Table 5). The lowest IC50 of the samples was

compared with that of the synthetic antioxidant BHT which indicated a higher IC50

10
in samples (Table 4). The difference between the IC50 of the samples and the

standard can be attributed to the differences in the type of phenolic compounds and

flavonoids.

The results showed that the total phenolic content in shoots resulted in the

highest amount when 0.9 kg per hectare humic acid was applied in combination

with 7.5 tons per hectare vermicompost. Accordingly, the average became 516.50

mg per 100 g dry weight (Table 5). An accurate and stable measurement of total

phenol content is merely not a very appropriate method to demonstrate the power

of the antioxidant capacity of the plant. Other characteristics are also involved in

defining the strength of high antioxidant activity in plants. These factors include

the type and amounts of individual compounds, phenol and flavonoid contents of

the plant material, which could be used as indicators of measurement. The total

phenol content, as measured by the Folin– Ciocalteu reagent (FCR), cannot be a

certain measure of the total phenol content in plants because the reaction between

the Folin’s reagent and phenolic compounds in extracts is nonspecific. In other

words, not only phenolic compounds in the extract react with Folin, but also other

substances in the extract could react with Folin, i.e. vitamins, amino acids,

carbohydrates, organic acids and aromatic amines (Salmanian et al., 2013 and

Mohsen & Ammar, 2009). It also seems that using gallic acid, as a standard, to

calculate the amount of total phenol content is not an exclusive indicator. Gallic

acid and hydroxybenzoic acids are also phenolic compounds but have a flavonoid

nature, whereas the mentioned method simply indicates the non-flavonoid

compounds. Considerable amounts of phenolic compounds in the plant are

flavonoid components such as flavones, flavonoids, caffeic acid, coumaric acid,

catechin, rutin, chlorogenic acid, and quercetin. In measuring the flavonoids, we

11
must also take notice that none of the colorimetric methods can detect all types of

flavonoids in the extract. For this reason, in this study, flavonoids such as flavones

and flavonols were identified and measured based on the aluminum chloride

colorimetric method. In other words, the aluminum chloride colorimetric method

can detect flavones and flavonols because they are the only components that can

form a stable complex with aluminum chloride (Salmanian et al., 2013 and Chang

et al., 2002).

Results also showed that the flavonoids in plants are strongly influenced by the

combined application of vermicompost and humic acid. The highest total content

of flavonoids was achieved by the treatment of 0.9 kg per hectare humic acid

combined with 7.5 tons per hectare vermicompost. The treatment yielded an

average of 817.77 mg/100 g DW (Table 5).

In this study, flavones and flavonols were significantly affected by the

combined use of humic acid and vermicompost. According to the results, the

highest content of flavones and flavonols were 188.04 mg/100 g DW, which

obtained by 0.9 kg per hectare humic acid with 10 tons per hectare vermicompost

(Table 5). The phenolic components in plant shoots showed that the gallic acid,

caffeic acid, p- coumaric acid and catechin are strongly influenced by the

combined effect of vermicompost and humic acid. The content of ellagic acid in

shoots was highest (with an average of 262.51 mg/100 g DW) when vermicompost

was used alone (5 tons per hectare). Also, the results showed that caffeic acid is the

dominant phenolic compound in chicory plant (Table 6). The presence of phenolic

acids in the extract of chicory confirmed the high antioxidant ability of this plant

(Table 7).

12
 Phenolic acids are one of the most important groups of secondary metabolites.

They are found in medicinal plants, and because of their carboxyl groups and

hydroxyl, they show strong antioxidant activities (Shahidi, 2000; Georgé et al.,

2005 and Michalak, 2006). Generally speaking, phenolic acids include two main

groups consisting of hydroxycinnamic acids and hydroxybenzoic acids. The

difference between these two groups is the methylation and hydroxylation patterns

in their aromatic ring. Hydroxycinnamic acids are similar to some other secondary

metabolites such as caffeic acid, chlorogenic acid, coumaric acid, and ferulic acid,

which have a side chain of propionic, instead of a carboxyl group in its structure,

and they normally show higher antioxidant activities than the hydroxy benzoic

acids such as gallic acid. Furthermore, the content of benzoic acid is usually much

lower than cinnamic acid in plants (Salmanian et al., 2013; Natella et al., 1999;

Vuorela et al., 2004; Mattila & Hellström, 2006 and Bondia-Pons et al., 2009).

Flavonoids such as flavones and flavonols have important effects on cell biology.

One of those effects is the removal and detoxification of free radicals which causes

damages to cells (Winkel-Shirley, 2002). The use of organic manufacturing

systems to increase the biosynthesis of flavonoids and flavonoid compounds in

plants has been effective, and vermicompost has had an important role in this

regard. Increased production of flavonoids in chamomile (Matricaria recutita L.)

(Vildova et al., 2006) as well as increased amounts of kaempferol and quercetin –

among flavonoids – have been successfully found in cultivated tomato (Mitchell et

al., 2007). Some other researchers also reported the positive influence of

vermicompost in enhancing the synthesis of flavonoids in plants (Kheiry et al,

2016).

13
 Various valuable organic and inorganic compounds contain humic acid. Other

compounds that can be absorbed by medicinal plants for their growth and yield to

enhance their quality include peptides, phenolic acids and esters, fatty acids and

oils, nucleic acids, monosaccharides and polysaccharides, alkanes, aldehydes,

tannins, amino acids and aromatic and aliphatic groups (Harper et al., 2000 and

Soleimani et al., 2012).

In a study by Anari Anaraki et al (2016) on pomegranate, the use of humic acid

caused the synthesis of phenolic compounds and flavonoids such as PAL, and the

amounts of these compounds increased in fruits treated with humic acid. This could

be explained by hormonal changes and the increased activity of enzymes involved

therein.

A positive correlation could be found between the amounts of phenolic

compounds and antioxidant power of the plant. In a similar study, a positive role of

humic acid was observed in increasing the total phenolics and antioxidant activity

of Calendula officinalis extract (Allahvirdizadeh and Nazari Deljou, 2014). The

usage of humic substances increases the plants’ ability to scavenge for free radicals

(Cacco et al., 2000) due to the complexity of secondary metabolites in plants,

thereby creating difficulties in a clear relationship between these compounds and

the antioxidant ability of plants (Zheng & Wang, 2001). Increased amounts of

phenols were observed in the broccoli after application of organic fertilizers. This

clarifies the role of such fertilizers in the biosynthesis of compounds that induce

the production of more shikimic acid, leading to a more proliferous production of

flavonoids and other phenolic compounds (Sousa et al., 2008 and Naguib et al.,

2012). The medicinal plant, Silybum marianum, was studied in a relevant context,

and it was suggested that the application of organic fertilizers increases phenolic

14
compounds significantly (Abdolah zareh et al., 2013 and Haj Seyed Hadi et al.,

2008). By animal fertilizers, the artichoke plant can increase its chlorogenic acid in

the bud (Emami Bistgani et al., 2015). Rooster et al. (1985) reported an increase in

phenolic derivatives, and approved it after the application of nitrogenous

compounds. It is worthwhile to mention that the application of nitrogenous

compounds increase the absorption of nitrogen. This reduces the amounts of

phenolic compounds in most cases. For example, the use of nitrogen fertilizer with

potassium and phosphorus on the Echinacea purpurea plant reduced its phenolic

compounds (Dufault et al., 2003). Results obtained by different researchers

indicate that the application of different amounts of varied elements is as important

as the type of elements. To note a few, the ratio of carbon to nitrogen in plants

stimulates the production of carbon-containing metabolites such as phenolic acids.

On the condition that this ratio is in favor of nitrogen, nitrogen-containing

compounds become evident by activation of the alkaloid synthesis pathway in the

plant (Pessarakli, 2001). The suitability of humic acid on increasing the phenolic

components over other organic acids has been demonstrated by previous reports

(Malik & Azam, 1985). Therefore, the ratio of carbon to nitrogen in organic

fertilizers – such as humic acid and vermicompost – can increase the plant’s

phenolic compounds, which ultimately results in a stronger antioxidant activity in

the plant. Humic substances in vermicompost have a role in plant health. These

substances increase the biosynthesis of phenolics such as flavonoids and

anthocyanins, and thus improve the quality of products and increase the resistance

of plants to stressful situations (Theunissen et al., 2010). The highest amounts of

flavonoids and other phenolic compounds in asparagus was obtained after the

application of vermicompost (Saikia & Upadhyaya, 2011). Moreover, the amounts

15
of phenolic compounds in leaves is higher than in any other organ, owing to

photosynthesis and the higher content of a precursor in the phenylpropanoids

production path (Davis et al., 2004). Plant antioxidants can prevent many diseases

such as cancer, cardiovascular disease, liver disorders, Alzheimer’s, diabetes and

inflammation. Therefore, increasing the antioxidant activity in plants could have a

key role in preventing human diseases (Uttara et al., 2009).

Conclusion

In this study, the highest dried material of chicory which is a criteria for herb

yield was achieved by application of 0.9 kg per hectare humic acid in combination

with 10 tons per hectare vermicompost. However, the highest total contents of

phenolics and flavonoids were achieved when humic acid was applied at 0.9

kg/hectare in addition to vermicompost at 7.5 ton/ hectare. The highest caffeic acid,

as a major phenolic component in chicory, was achieved by treatments of 0.3 kg

per hectare humic acid combined with 5 tons per hectare vermicompost. Moreover,

the content of ellagic acid was highest when vermicompost was applied alone, by 5

tons per hectare.

Acknowledgments

We thank Shiraz University Vice Chancellor for funding this research.

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Table 1. Results of soil sample analysis in cultivation area

mg/l (ppm) mg/kg E Prof

soil Sa Si Cl Cl S
N OC p C ile
text nd lt ay Me N M C C M Z P
Ni Fe K P % % H ds/ (cm
ure % % % q/l a g a u n n %
m )

Silty 4
10. 4 45. 0. 6. 1 1 0. 9. 3. 1 0. 1.4 7. 1.9 35
- 1.5 8 5 0-30
72 4 28 38 78 6 2 56 34 18 4 09 9 82 8 .6
clay 0

28
 Table 2. Components analysis of vermicompost used in the study (prepared from agricultural station

of shiraz university)

mg/l (ppm) mg/kg %

EC ds/m C/N
Mo Fe Na Mg Ca Cu Mn Zn P K N pH

40 5000 0.8 0.9 2.73 20 276 110 0.4 0.4 1.6 7.4 1.2 21

Table 3. Vermicompost fertilizer recommendation for plants to determine the optimal concentration

according to soil test results

120 kg nitrogen per

Recommended amount of nitrogen in soil organic matter which is below 1.49%
hectare

recommendation on soil phosphorus application to which the phosphorus is 100 kg P2O5 use

measured by Watanabe and Olsen (1965) method (14 mg/kg) per hectar

Recommended amount of potassium in the target soil to which the potassium

0 kg K2O per hectar
levels is higher than ppm 201 (450 ppm)

Expected yield 20 ton per hectar

The optimal amount of vermicompost necessary in soil after calculations and

conversions the recommended amounts of chemical fertilizers to organic 7.5 ton per hectar

manure

29
Table 4. Comparing the lowest IC50 of the samples with synthetic antioxidant BHT

IC50 (mg DW/1ml 70% methanol)

The lowest IC50 measured in the laboratory 0.1003

The measured IC50 synthetic antioxidant BHT (Standard) 0.0407

Table 5. Biomass production, total phenolic and flavonoids contents of chicory as affected by humic acid and vermicompost

treatments

(g/plant) (mg/100g DW)

humic Inhibition of IC50 (mg Total Flavones Total

Vermicompost Fresh Dry
acid free radicals DW/1ml 70% flavonoids and phenol
(Ton/ha) weight weight
(Kg/ha) (%) methanol) flavonols

58.94 ± 5.947 ± 65.47 ± 4.05 0.169 ± 0.00 83.16 ± 62.57 ± 35.99 ±

0
2.08 j* 0.04 f efg a 5.04 k 14.47 g 2.85 j
81.56 ± 7.45 ± 60.77 ± 2.43 0.1633 ± 0.00 76.08 ± 82.45 ± 38.87 ±
0.3
1.68 hi 0.53 ef g ab 2.68 k 6.88 fg 1.85 j
0 10.92 ±
92.84 ± 60.19 ± 3.69 0.1586 ± 0.00 227 ± 71.05 ± 110.57
0.6 1.94
2.79 fg g ab 12.58 g 11.56 fg ± 7.86 h
bcd
82.28 ± 11.56 ± 64.96 ± 0.69 0.149 ± 0.00 484.21 ± 124.17 ± 184.98
0.9
1.12 hi 0.18 bc efg bcd 12.08 e 8.18 d ± 5.03 g
76.04 ± 7.20 ± 62.37 ± 3.24 0.163 ± 0.00 108.81 ± 63.28 ± 32.19 ±
0
2.62 hi 0.3 ef g ab 9.78 jk 6.39 g 1.91 j
86.17 ± 12.03 ± 63.90 ± 4.76 0.156 ± 0.00 146.52 ± 110.41 ± 80 ±
0.3
3.17 gh 1.48 cd fg abc 11.54 i 6.64 de 4.22 i
10.82 ±
5 118.54 69.10 ± 2.23 0.143 ± 0.00 328.42 ± 122.87 ± 237.6 ±
0.6 0.34
± 3.79 e defg cde 11.32 f 4.84 d 5.17 e
bcd
10.75 ±
120.21 73.88 ± 2.8 0.137 ± 0.00 560.84 ± 157.9 ± 251.96
0.9 1.36
± 1.55 e bcde def 7.38 d 7.19 bc ± 3.81 e
bcd

30
 80.92 ± 7.98 ± 61.26 ± 0.59 0.1613 ± 0.00 133.95 ± 69.78 ± 46.03 ±
0
1.86 hi 0.52 def g ab 3.78 ij 2.56 fg 2.92 j
8.97 ±
98.22 ± 76.48 ± 2.46 0.1333 ± 0.00 191.76 ± 134.98 ± 118.53
0.3 0.31
2.58 f bcd ef 8.12 h 5.85 cd ± 5.21 h
7.5 cde
195.74 18.35 ± 78.10 ± 1.64 0.1233 ± 0.00 514.49 ± 163.44 ± 423.56
0.6
± 4.26 a 0.68 ab bc f 10 e 17.26 ab ± 2.72 c
180.55 17.98 ± 87.37 ± 0.35 0.1013 ± 0.01 817.77 ± 172.75 ± 516.5 ±
0.9
± 2.01 b 0.17 a a g 9.25 a 8.58 ab 21.34 a
73.28 ± 11.10 ± 72.60 ± 4.15 0.1526 ± 0.00 148.3 ± 90.82 ± 43.7 ±
0
1.53 i 1.15 bc cdef bc 5.56 i 7.55 ef 2.68 j
129.79 13.45 ± 79.90 ± 0.97 353.95 ± 152.35 ± 206.22
0.3 0.127 ± 0.00 f
± 4.26 d 1.73 b abc 13.45 f 4.8 bc ± 9.8 f
10
158.69 17.54 ± 87.21 ± 1.96 0.1003 ± 0.01 593.41 ± 186.7 ± 305.70
0.6
± 3.54 c 0.95 a a g 25.47 c 6.31 a ± 3.98 d
200.07 20.29 ± 81.93 ± 4.06 0.104 ± 0.01 727.24 ± 188.04 ± 479.8 ±
0.9
± 7.09 a 1.03 a ab g 13.79 b 2.36 a 4.77 b
Data are means (± standard error) of three replicates (n = 3). *For each factor and in each column, means followed by the

same letter(s), are not significantly different (p < 0.05) based on Duncan's Multiple Range test.

Table 6. Phenolic compounds variation of chicory under different humic acid and vermicompost treatments

(mg/100g DW)

vermicompost humic acid Ellagic p- Coumaric

Gallic acid Caffeic acid Catechin
(Ton/ha) (Kg/ha) acid acid

38.3 ± 3.73 431.1 ± 1.75 201.26 ± 124.46 ± 139.34 ± 2.22

0
d h 1.91 c 2.35 ef g
45.35 ± 583.4 ± 1.83 191.59 ± 124.16 ± 169.34 ± 3.56
0.3
2.04 d gh 13.3 c 8.22 ef g
0
682.46 ± 897.6 ± 11.67 ± 106.97 ± 898.8 ± 85.18
0.6
38.07 b 126.24 ef 0.71 e 1.83 fg bcd

31
 790.83 ± 1236.1 ± 6.88 ± 198.89 ± 893.21 ± 3.53
0.9
3.84 ab 105.57 bc 0.59 e 1.62 bc cd
41.84 ± 583.6 ± 262.51 ± 152.52 ± 180.13 ±
0
1.97 d 59.95 gh 4.23 a 20.09 de 12.11 g
349.77 ± 2773.1 ± 35.67 ± 187.13 ± 696.5 ± 4.87
0.3
2.65 c 60.23 a 1.22 d 12.24 bc ef
775 ± 65.63 1171.4 ± 10.578 ± 262.74 ± 732.26 ± 18.9
5 0.6
ab 174.91 bcd 3.72 e 2.26 a ef
751.05 ± 1074 ± 11.34 ± 173.54 ± 809.27 ±
0.9
36.88 ab 149.01 cde 3.48 e 4.85 cd 11.51 de
45.97 ± 760.5 ± 233.38 ± 171.69 ± 198.56 ±
0
0.32 d 13.73 fg 2.7 b 1.84 cd 13.05 g
684.02 ± 974.2 ± 5.03 19.40 ± 151.2 ± 1.75 1024.36 ±
0.3
64.2 b def 1.56 e de 4.49 ab
846.77 ± 1202.7 ± 46 7.84 ± 961.95 ±
7.5 0.6 92.54 ± 1.2 g
9.64 a bcd 0.53 e 42.43 abc
713.01 ± 1153.3 ± 2.2 6.73 ± 109.35 ± 1036.51 ±
0.9
6.86 b bcd 0.97 e 0.00 fg 17.53 a
765.64 ± 1113.3 ± 7.14 ± 124.34 ± 963.21 ± 9.44
0
38.92 ab 69.39 cde 0.18 e 26.36 ef abc
787.08 ± 1044.3 ± 7.17 ± 121.4 ± 5.39 896.59 ±
0.3
71.14 ab 92.28 cde 0.74 e efg 116.41 bcd
777.92 ± 1233.8 ± 4.5 6.93 ± 106.88 ± 679.84 ± 3.66
10 0.6
41.47 ab bc 0.64 e 3.97 fg f
831.87 ± 1373.9 ± 10.67 ± 204.75 ± 866.37 ± 7.98
0.9
17.75 a 44.12 b 1.48 e 5.82 b cd

Data are means (± standard error) of three replicates (n = 3). For each factor and in each column, means followed by the

same letter(s), are not significantly different (p < 0.05) based on Duncan's Multiple Range test.

Table 7. Correlation among measured factors of chicory influenced by organic fertilizers

fresh dry
aeria aeria flavone
total total Galli p-
l l IC5 s and caffei ellagi catechi
%I flavonoi pheno c coumari
parts parts 0 flavonol c acid c acid n
ds l acid c acid
yield yield s
s s
Fresh 0.88 0.8 - 0.95 0.61 0.21 -0.53
1 0.85 ** 0.84 ** -0.11 ns 0.51 **
aerial ** 2 0.8 ** ** ns **

32
 parts ** 4
yields **
Dry -
0.7
aerial 0.7 0.86 0.67 0.39 -0.63
1 2 0.81 ** 0.8 ** -0.1 ns 0.62 **
parts 9 ** ** ** **
**
yields **
-
0.8 0.81 0.57 0.16 -0.52
%I 1 0.77 ** 0.8 ** -0.2 ns 0.5 **
5 ** ** ns **
**
-0.84 -0.64 -0.22 0.59 -0.55
IC50 1 -0.83 ** -0.84 ** 0.11 ns
** ** ns ** **
Total
0.94 0.68 0.21 -0.61
flavonoi 1 0.84 ** 0.01 ns 0.57 **
** ** ns **
ds
flavones
0.84 0.72 0.34 -0.68 -0.001
and 1 0.61 **
** ** * ** ns
flavonols
Total 0.65 0.21 -0.58
1 -0.02 ns 0.57 **
phenol ** ns **
Gallic 0.33 -0.93
1 0.05 ns 0.89 **
acid * **
Caffeic -0.50
1 0.29 * 0.4 **
acid **
Ellagic -0.92
1 -0.05 ns
acid **
p-
-0.03
Coumari 1
ns
c acid
Catechin 1

ns, * and **: Indicate non-significance and significance at 5 and 1% probability levels, respectively.

33