25. evaluation of major determinants.full

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International Journal of Agricultural

Science and Research (IJASR)

ISSN 2250-0057

Vol. 3, Issue 3, Aug 2013, 197-210

TJPRC Pvt. Ltd.

EVALUATION OF MAJOR DETERMINANTS IN SOIL NUTRIENTS AMELIORATE FOR

PRODUCTION OF CANCER CHEMOPREVENTIVE AGENT CURCUMIN IN RHIZOMES

OF CURCUMA LONGA L.

AKHILESHWAR KUMAR SRIVASTAVA1, RAGHVENDRA RAMAN MISHRA

2, MALLIKA TEWARI

3,

HARI SHANKAR SHUKLA4

& BIJOY KRISHNA ROY5

1,5Department of Botany, Banaras Hindu University, Varanasi, Uttar Pradesh, India

2,3,4Department of Surgical Oncology, Banaras Hindu University, Varanasi, Uttar Pradesh, India

ABSTRACT

The response to soil nutrients on the production of curcumin (cancer chemopreventive agent) in turmeric rhizomes

was studied. Four different sampling sites in northern-India with variable soil properties were selected. Curcumin

production in relation to soil physico-chemical properties was assessed. The variations in curcumin amount depended on

soil pH, level of nutrients (Ca, Cu, Fe, Mg, Zn, N, P, K), and organic C in soil. The curcumin amount in turmeric from

Varanasi district (9.03%) was highest followed by site at Darbhanga (6.04%), Mirzapur (4.47%), and Allahabad (4.43%).

The results revealed that curcumin content enhanced corresponding to the favourable pH (7.1) and high nutrient levels in

soil. Among the soil nutrients, N, K, and total NPK showed positive and significant correlation with curcumin production.

The level of N, K, and combined NPK in the soil were the major determinants of curcumin production.

KEYWORDS: Turmeric, Curcumin, Nutrients, HPLC, Physico-Chemical Properties of Soil, Correlation Coefficient

INTRODUCTION

India has been the great repository of medicinal plants since long (Chattopadhyay et al., 2004). Turmeric

(Curcuma longa) is broadly used in disciplines of Ayurveda, Unani and Siddha (Ammon et al., 1991; Eigneret al., 1999).

The plant belongs to family Zingiberaceae, and originally reported from South and East India and Indonesia.

Now-a-days, India is the largest turmeric producer, exporter, and consumer in the world (Kamble et al., 2011). Presently,

turmeric has been used in many countries as the most favourite spice (Ammon et al., 1991; Ishimine et al., 2004).

Nonetheless, curcumin and volatile oils in the rhizome have proved anti-inflammatory, antimutagen, anticancerous,

antibacterial, anti-oxidant, anti-fungal, antiparasitic, and detox properties (Ammon et al., 1991; Osawa et al., 1995). Some

other active ingredients of turmeric also enhance the function of liver and kidney, and reduce biliary, diabetic and hepatic

disorders (Ammon et al., 1991). Besides above, anticancerous properties of curcumin have been proven experimentally on

different cell lines and animals with different levels of carcinogenesis in skin, breast and gastrointestinal tracks (Rao et al.,

1995; Huang et al., 1994; Huang et al., 1998; Aggarwal et al., 2003).

Looking at spread of cancer and the implication of turmeric as the remedial measure, emphasis has been on high

production of turmeric. There is a lack of information on the agronomic aspects of turmeric production in general, and the

weather relationship in particular. The report on status of scientific agronomic practices for turmeric farming is also

meagre relative to other turmeric producing countries.

Many factors that play vital role in growth/development of turmeric rhizome and leaves are nutrition, cultivation

practices, genotype, and the environment factors (Gregory, 1988; Loomis and Amthor, 1999; Azam-Ali and Squire, 2002).


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198 Akhileshwar Kumar Srivastava, Raghvendra Raman Mishra,Mallika Tewari, Hari Shankar Shukla & Bijoy Krishna Roy

Besides above, growth yield and the quality of cultivars also depend on the soil type, and its nutrients levels (Oya, 1972;

Oya et al., 1977). Therefore, it seems imperative to look for the type of soil and the levels of nutrients for high yield

cultivars (Oya, 1972). Kandiannan et al. (2002) reported thatplant requires an annual average rainfall (640 to 4200 mm)

and the annual mean temperature of 18.2 to 27.4

0

C. However, the requirement of optimum temperature is in 25-35

0

Crange for sprouting of turmeric rhizome. Although the luxuriant growth of turmeric occurs in shades, larger rhizomes are

produced in the open field exposed to sunlight (Ridley, 1912).

Many agronomists earlier employed correlation path and cluster analyses to study the soil type that favoured high

quality turmeric yield (Chenchaiah et al., 2002; Sarma et al., 2003; Nybe et al., 2008) based on the rhizome yield and soil

factors, but failed to explain the uptake of soil nutrients that favours plant growth/metabolism. Among the metabolites,

curcumin is the important one in turmeric rhizome, and its amount increased significantly under Fe deficiency, and it also,

reciprocates with the enhanced levels of soil organic acids (Dixit and Srivastava, 2000). Looking at the medicinal potential

and the numerous pharmaceutical aspects of curcumin, it seems imperative to increase its yield and also to develop the

desired turmeric traits.

In agricultural fields the elimination of soil micronutrients leads to soil deficiency in many countries. The

deficiency of iron is most common to calcareous or alkaline soils. The application of N, P, and K fertilisers at higher doses

also decreased the curcumin amount in rhizomes, but the graded dose of N, P, and K increased (Rao et al., 1975;

Mohanbabu and Muthuswamy, 1984; Ahmed et al., 1988; Meenakshi et al., 2001). Therefore, we attempted to study the

distribution pattern of soil micronutrients levels in North-India keeping in views the possible improvement of turmeric

cultivation, and also the curcumin contents.

Curcumin is easily extracted from the turmeric rhizome by the solvent extraction method, and its amount analysed

by using HPLC (Balashanmugan, 1991). The study was conducted to determine the rhizome curcumin content in relation

to soil organic and inorganic nutrients levels in soils from different locations of North-India.

MATERIAL AND METHODS

Study Site

The turmeric rhizomes were collected from four different locations at district of Varanasi, Allahabad, Mirzapur

(Uttar Pradesh) and Darbhanga (Bihar). All the locations display different geographical status and soils properties.

Varanasi is located in the eastern part of the state of Uttar Pradesh (82 56E - 83 03E longitude and 25 14N -

25 23.5N latitude) in the middle Ganges valley of north India. This site is enriched with the fertile gangetic alluvial soil.

Allahabad in the southern part of Uttar Pradesh state, (2526 N latitude and 8150 E, longitude), stands at the confluence

of the river Ganga and Yamuna with the intermediate alluvial soil. Mirzapur is located between 25.15N latitude and

82.58E longitude in the southern part of the state dominated by the Vindhyan alluvial soil. Darbhanga is located in the

north Bihar (26.17N latitude and 85.9E longitude) enriched with calcareous and alluvial soil.

Sample Collection

Soils samples are collected randomly at 5 locations of each site at 10 meter intervals from 0-10 cm depth and kept

separately. All the samples were brought to the laboratory and dried at 800C, and 100 g of the dried soil was sieved (mesh,

2 mm).

The turmeric rhizomes were collected from the agricultural field of each location, and dried at 500C (48 h) and

powdered by grinding for HPLC analysis.


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Evaluation of Major Determinants in Soil Nutrients Ameliorate for Production of 199Cancer Chemopreventive Agent Curcumin in Rhizomes ofCurcuma longaL.

Determination of Soil pH

Soil pH was determined in the 1:1 soil-water mixture using the glass electrode (Peech, 1965).

Determination of Nutrients Concentration

Metal Analysis

Soil (1.0 gm) sample was placed in beaker and added with 7.0 ml ternary acid (HNO3:H2SO4:HClO4; 5:1:1), and

incubated (10-12 h). After digestion, the samples were diluted in 50 ml double distilled water and filtered. Metal

concentration in samples as analysed by Atomic Absorption Spectrophotometer (Perkin Elmer model 2380) against

standard metal solutions. Metal concentration is expressed in g/g soil dry wt.

N, P, and K Analysis

The nutrients N, P, K, were determined separately for N by micro-Kjeldahl procedure (Peach and Tracey 1956), P

was measured after digestion with ternary acid (HNO3/H2SO4/HClO4; 10/1/4, V/V) and assayed by phosphomolybdic acidblue assay (Jackson, 1958). K was determined by Flame photometer (Mediflame 127) following the method of Jackson

(1958).

Estimation of Organic Carbon

Soil organic carbon was determined by Walkley and Blacks rapid titration method (Jackson, 1958). Air-dried soil

sample (1.0 g) was taken in 500 ml Erlenmeyer flask and added with K2Cr2O7 (10 ml) and H2SO4 (20 ml). The mixture was

swirled until soil and reagents fully mixed. After 30 min, 200 ml of double distilled water was added. Organic carbon was

determined by titration with 0.5N FeSO4 solution containing 3-4 drops of O-phenapthroline as the indicator. Organic

carbon was calculated as:

Organic carbon = (Meq K2Cr2O7-Meq FeSO4) 0.003 100 f / weight of soil (gm)

Where f = 1.33

HPLC Analysis

The HPLC model, HP C-18 column (1250 4.6 mm), and Millipore (0.454 m) membrane filter was used for

filtration. The injection system used 20 l sample loop. The solvents (methanol and acetonitrile) were from Spectrochem

laboratories (Mumbai, India). Water for HPLC was purified by the Milli Q water purification (Synergy, Bangalore, India).

The curcumin standard was purchased from MP Biomedicals, LLC BP 50067, Illkirch, France.

Curcumin from turmeric powder was extracted solvent extraction method(ASTA,1958). About 10 mg standard

curcumin sample was taken in 100 ml volumetric flask and diluted up to 10 ml by methanol and kept as stock. The extract

(2ml) was made up to 10 ml by adding of methanol. Similarly, 10 mg of each turmeric powder sample was put in 100 ml

volumetric flasks and added with 10 ml methanol. After filtration, the extract (2 ml) was transferred to the volumetric flask

and diluted to 10 ml with methanol.

For the separation and detection of the curcumin, a chromatographic system consisted Water (Malaysia) Model

2998 (USA), with the plus autosampler (USA). All the solvents were filtered using 0.45 m membrane filter (Millipore,

USA) before injection in HPLC. The standard curcumin (20 l) and other turmeric samples were subjected to HPLCanalysis. The separation and elution were performed by solvents (methanol 24.1%, acetonotrile 38.2% and deionised water

37.7%) as the mobile phase with the flow rate of 1 ml min-1

at 350C, and detected at 425 nm and chromatograms were

processed by Empower 2 software.


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The concentration of curcumin in different samples was determined by comparing with the standard curcumin

(Figure 3). The amount of curcumin in different turmeric samples was calculated by using the following equation (Kamble

et al., 2011).

Cs = As Cc 100 / Ac

Where Cs = concentration of curcumin in turmeric sample (g/ml) Cc = concentration of standard curcumin

(g/ml), As = area of sample, AU/min and Ac = area of standard curcumin, AU/min. The amount of curcumin in turmeric

sample from each site was determined and expressed in percent values.

Statistical Analysis

Statistical analysis was performed using the SPSS version 16.0 software (IBM, USA), and the mean and standard

error analysed by T - test. Analysis of mean of replicate of each sample was performed to go for the accuracy and

comparing the results of all the sampling sites. The correlation of variable data was obtained to establish the possible

correlation between production of curcumin and soil physico-chemical properties.

RESULTS

Physicochemical Properties of Soil

Table 1 shows the physico-chemical characteristics of soils from different sites. The level of nutrients varied

among the samples but their levels in the soils from Varanasi favoured maximum curcumin production relative to other

sites, Darbhanga, Mirzapur, or Allahabad. The soils pH varied from 8.5-6.8 and was highest (8.5) in soils of Mirzapur and

the lowest pH (6.8) for Allahabad, followed by soils from Varanasi (pH, 7.1).

Soil Nutrients Content

The concentration of Zn was highest in soils of Allahabad, whereas the amount of Cu, Ca, and Mg was highest in

Darbhanga soils compared to other sites. Similarly, the maximum Fe concentration (65.80 g/g) was found in soils of

Mirzapur. The Cu concentration (1.10 g/g) was lowest in soils of Allahabad relative to soils of different location.

Distribution pattern level of Ca was 38.84 g/g, 47.05 g/g, 32.53 g/g, and 35.64 g/g in soils of Varanasi, Darbhanga,

Allahabad, and Mirzapur, respectively, and indicated that the element was maximum among the other metals in soils. In

comparison, Cu concentration was lowest in soils of all the sites.

Level of NPK and Organic Carbon in Soil

The amount of N, P, and K varied among soils of the sampling site (Table 1). The highest amount of N (41.21

ppm) and K (45.40 ppm) was observed for soils of Varanasi whereas their amount as comparatively low in soils of other

sites. The amount of P was highest (2.50 ppm) in soils of Darbhanga, and the lowest (1.70 ppm) for Allahabad. For

agricultural practices, N, P, and K were used in combination, and hence the level of combined NPK was the highest (88.51

ppm) in soils of Varanasi followed by Darbhanga > Mirzapur > Allahabad in the decreasing order (Figure 1).

The highest C level (0.61%) was observed in soils of Varanasi, and the lowest (0.26%) for Darbhanga (Table 1).

Curcumin Content in Rhizomes

Figure 2 compares the chromatograms of standard curcumin (2a) and turmeric samples 2(b1-b4). The curcuminwas detected at 425 nm and compared with retention time (8.3 min) of the standard. The highest peak of curcumin was

observed in samples of Varanasi, followed by the smallest one in samples of Allahabad. The levels of curcumin in samples

of Allahabad and Mirzapur were comparable. Figure 3 shows a graphical presentation of curcumin amount in turmeric


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rhizomes. Curcumin contents in samples varied among the rhizomes corresponding to each soil. The highest curcumin

content (9.03%) was from Varanasi, followed by 6.04% from Darbhanga, 4.47% from Mirzapur, and the lowest (4.43%)

from Allahabad, respectively.

The correlation coefficient was used to establish the correlation of curcumin production witrh pH and nutrient

contents in soils as shown [Figure 4(a-e)]. The amount of curcumin was positively correlated with N (R = 0.76) at P

0.05, K (R = 0.89) at P 0.01, and NPK (R = 0.83) at P 0.05. The results revealed that curcumin content was

significantly correlated with the level of N, K and combined NPK,whereas rest of soil nutrients were not significant

relation

DISCUSSIONS

In agricultural practices, the correlation between the soil factors and rhizome yield and metabolites synthesis has

been established (Li et al., 1999). The elevated level of micronutrients in soils also play significant role(s) in increasing

curcumin production depending on the absorption, accumulation and their role in the metabolic processes. The knowledge

about the agronomical aspects of turmeric and curcumin production is almost lacking, except for a few reports on the

cultivation of turmeric (Ishamin et al., 2004; Hossain et al., 2005). The supply and absorption of elements needed for

growth and metabolism and their participation with the metabolite production, are closely related. Here, on the basis of our

findings the level of organic and inorganic elements in soils and their role in synthesis of curcumin in rhizome has been

ascertained. The data on the evaluation of the soil physico-chemicals properties (pH, inorganic, and organic elements) in

soils from different locations of North-India have been incorporated in Table 1.

The rhizome curcumin content at four different sites of North-India ranged from 4.43 to 9.03% (Figure 3). Such

variations could be attributed to the difference in the soil physico-chemical properties. The maximum curcumin content

(9.03%) was from Varanasi soils and the lowest (4.43%) from Allahabad. Such findings support the earlier report on

curcumin content in rhizomes of commercially produced turmeric where in the value ranged from 2 to 8% (Heath et al.,

2004). In a previous study, no direct correlation between rhizome growth and the level of micronutrients K, Ca, and Mg in

soils has been reported (Oya, 1972).

Effect of pH Soil on Curcumin Content

The pH levels also influence soil fertility in respect of growth and yield of different plants. Earlier, the pH of soil

(4.5-7.5) has been considered suitable for better growth as well as the enhancement of curcumin production in the turmeric

rhizome (Pradhan et al., 2003; Singh and Rai, 2003). Results incorporated in Table 1, indicate thatsoil pH varied from 6.8

at Allahabad to the maximum of 8.5 at Mirzapur. The curcumin amount in the rhizome ranged from maximum of 9.09% to

the minimum of 4.43% as shown in Figure 3. Here, it is of great concern that alkaline pH (8.5) at Mirzapur soil favoured

higher curcumin amount in comparison to samples from Allahabad. Although, the data in Figure 4(a) indicated the

correlation coefficient of soil pH (R = 0.109) at P 0.05 in relation to the curcumin content, it was not significantly related

indicating that not only the individual soil factor but also the soil pH, organic/inorganic elements were playing the

synergistic role(s) on curcumin production.

Relation of Soil Nutrients on Production of Curcumin

The soil metalconcentration varied at different locations as shown in Table 1. It is evident that the amount ofcurcumin in rhizomes enhanced with the certain level of particular element in the soil. The concentration of Ca was

dominant in soils of Varanasi, Darbhanga, while Fe was maximum (65.81 g/g dry wt) for soils from Allahabad. The Ca

concentration decreased in the order: Darbhanga 47.05 > Varanasi 38.84 > Mirzapur 35.64 > Allahabad 32.53 g/g dry wt


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followed by Cu (Darbhanga 2.79 > Varanasi 2.54 > Mirzapur 1.50 > Allahabad 1.10 g/g dry wt), respectively. According

to the present data, the range of curcumin amount can be arranged as: Varanasi 9.03 % > Darbhanga 6.04% > Mirzapur

4.47% > Allahabad 4.43%. Figure 4(b.1-b.5) showed correlation coefficient of soil metal concentration at P 0.05 with no

relationship with curcumin production. The result also indicated that single nutrient at dominant level in the soil, has nopositive role in enhancing the curcumin production. In the case of the Mirzapur iron-enriched soils, the amount of

curcumin was lower in comparison to Varanasi and Darbhanga, but higher than that of Allahabad. Figure 4(b.3) shows that

the correlation coefficient of Fe (R = 0.164) at P 0.05 in response to curcumin content contradicts the view of Dixit and

Srivastava (2000) on high curcumin production in the iron-deficient soils. Presently, the enrichment and deficient status of

individual metal in the soil did not count much in curcumin production.

Impact of N, P, and K on Productivity of Curcumin

Figure 4(c.1 and c.3) showed the correlation coefficient for N (R = 0.767) at P 0.05 and K (R = 0.894) at P

0.01 implying that these significantly influenced the curcumin contents, and supported the earlier finding (Hikaru et al.,2007). Earlier, Singh et al., (1998) reported that increasing the level of K in soils had positive effect on growth of

rhizomes. In comparison, the values in Figure 4(c.3) also had high correlations with curcumin production. However, Figure

4(c.2) shows that insignificant correlation coefficient of fertilizer P (R = 0.0449) at P 0.05, and indicated that P

individually might not be playing significant role in curcumin production.

Effect of Total Combined NPK

In most parts of India, usually NPK are used as the fertilisers to improve crop growth/yield. The relation between

combined NPK level and curcumin production was correlated (coefficient correlation R = 0.83) at P 0.05), and expressed

in Figure 4(d) which is suggested that combined NPK levels in soils may be the suitable dose for enhancing the curcumin

amount. Highest amount of curcumin (9.03%) was from Varanasi, indicating that the combined action of NPK in

agricultural soils at suitable doses, was optimum. The present study showed that the combined NPK level and amount of

curcumin can be arranged as: Varanasi Darbhanga Mirzapur Allahabad (Figure 1 and 3).

Effect of Soil Organic Carbon on Curcumin Content

The level oforganic carbon seems helpful in increasing the soil capacity to maintain the micronutrient availability

balance between soil and the plant and thus improving the crop quality. Lal (2004) also reported that quality micronutrients

enhanced biomass production through supply of higher amount of organic carbon. In present finding, the highest organic

carbon concentration (0.61%) could be the factor for retaining the nutrients in soils of Varanasi, and might have resulted in

the maximum curcumin production. However, findings on the lower concentration of organic carbon (0.26%) in soils of

Darbhanga and increased production of curcumin (6.04%) in comparison with other two sites at Mirzapur, and Allahabad

with 0.35% and 0.27% of organic carbon in soils (Table 1) and their correlation coefficient (R = 0.74) at P 0.05 in

relation to curcumin production was insignificant as shown in Figure 4(e) and contradicted this assumption. This could be

explained that other soil factors or the synergistic effects of organic and inorganic nutrients might be playing a role in

enhancing curcumin production.

CONCLUSIONS

It is concluded that curcumin production was dependent mainly on the distribution of fertilizers N, K, or its

combination (NPK) in addition to certain levels of soil nutrients. Curcumin production corresponding to different locations

of North-India indicated that level of K in soil was playing more significant role. However, N and the combined NPK level


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in soils at suitable doses, may also play a positive role in the enhancement of curcumin production. These findings could

also added to agronomic practices for future line of action.

ACKNOWLEDGEMENTS

Author would like to express their gratitude for financial support from University of Grant Commission and to

Department of Botany, Banaras Hindu University, India for providing the instrumental facility for detection of metals in

soil samples and chromatogram of curcumin in different sample of turmeric rhizomes.

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APPENDICES

Table 1: Physico-Chemical Properties of Soil Samples Collected from Different Sites

Concentration of Metals g/g Dry wt. Amount of NPK in ppmOrganic

Matter in %Sites of

SamplingpH Zn Cu Fe Ca Mg N P K C

Varanasi 7.1 9.090.053 2.540.058 29.850.55 38.840.88 24.210.63 41.211.32 1.900.09 45.400.51 0.610.016

Darbhanga 7.8 6.280.036 2.790.059 20.050.34 47.051.53 28.740.87 39.500.66 2.500.13 35.340.75 0.260.010

Mirzapur 8.5 8.350.061 1.500.034 65.810.65 35.640.57 20.200.52 27.830.81 1.800.09 21.200.93 0.350.013

Allahabad 6.8 10.420.74 1.100.029 28.620.62 32.530.78 27.310.59 28.330.89 1.700.11 15.450.74 0.270.017

Figure 1: Total Concentration of NPK in Soils of Different Sampling Sites.

Results Shown as Means and Standard Errors. For Sampling Sites n=5


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2(a)

2(b.1)

2(b.2)

2(b.3)


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2(b.4)

Figure 2: Chromatogram of (a) Curcumin Standard and Curcumin in Rhizomes Samples from Different

Sites of North-India (b.1) Varanasi (b.2) Darbhanga (b.3) Mirzapur (b.4) Allahabad. The X-Axis Representsthe Retention Time of Separated Components. The Y-Axis Represents the Absorption Unit of Spectra

Figure 3: Variation of Curcumin Content in Rhizomes from Different Sites. Values Expressed as Percent of

Curcumin. Results Presented as Means and Standard Errors. For Samples of Turmeric Rhizomes n=5

4(a)


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4(b.1)

4(b.2)

4(b.3)

4(b.4)


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4(b.5)

4(c.1)

4(c.2)


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4(c.3)

4(d)

4(e)

Figure 4: Expresses Relation between Curcumin Contents with (a) pH of Soil (b.1-b.5) Concentration

of Metals Zn, Cu, Fe, Ca, and Mg in g/g Dry wt., Respectively (c.1-c.3) Amount of Fertilizers

in ppm (d) Level of Total NPK in ppm (e) Level of Organic C in Percent

25. evaluation of major determinants.full

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