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Accumulation of secondary metabolites in response to antioxidant activity of turmeric rhizomes co-inoculated with native arbuscular mycorrhizal fungi and plant growth promoting rhizobacteria
Scientia Horticulturae 204 (2016) 179–184
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Accumulation of secondary metabolites in response to antioxidant activity of turmeric rhizomes co-inoculated with native arbuscular mycorrhizal fungi and plant growth promoting rhizobacteria Shanti Chaya Dutta ∗ , Bijoy Neog Dept. of Life Sciences, Dibrugarh University, Dibrugarh, Assam, India
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Article history: Received 14 December 2015 Received in revised form 16 March 2016 Accepted 22 March 2016 Available online 18 April 2016 Keywords: Curcuma longa L. Bio-inoculants Antioxidant activity Flavonoids Phenolic contents Curcumin content
a b s t r a c t The present study was aimed at investigating the therapeutic potential of methanol crude extract of turmeric (Curcuma longa L.) rhizome co-inoculated with native arbuscular mycorrhizal fungi (Glomus, Gigaspora and Acaulospora sp.), phosphate solubilizer Bacillus megaterium and diazotrophic bacteria, Azospirillum amazonense and Azotobacter sp. To achieve the goals, parameters like free-radical (DPPH, ABTS) scavenging capacity, total phenolic contents, total flavonoids contents and percentage of curcumin content were examined. A significant increase of these secondary metabolites were observed in inoculated plants compared to non-inoculated controls revealing strong antioxidant activities of turmeric against DPPH and ABTS radicals in the range of 80–97%. The high amount of flavonoids (179.07–493.15 mg RE g−1 ), phenolic contents (83.07–151.54 mg GAE g−1 ) and percentage of curcumin (4.81–6.09%) present in all the methanolic extracts of dried rhizomes might be responsible for the observed antioxidant activity. © 2016 Elsevier B.V. All rights reserved.
1. Introduction A majority of human food crops are highly mycorrhizal depicting the ubiquitous nature of arbuscular mycorrhizal fungi (AMF) and the ameliorative effects of these fungi may result into some “healthier” food, either by having more concentrated nutrients (biofertilizer effect) or higher concentrations of defense-related compounds (bio-protective effect) (Antunes et al., 2012). Recently, secondary metabolites of plants which play a bioprotector role with taste and health related properties (Schreiner et al., 2012) are gaining much attention from the researchers. In this context, AMF inoculation could be an important strategy in biofortification of crops (Antunes et al., 2011). But, evidence to support their effectiveness is currently confounded by inadequate quality standards and insufficient knowledge of the underlying mechanisms, which have led to contradicting reports on field performance (Owen et al., 2015). There is, however, scope to engineer specific bioinoculant formulae like AMF, either the native ones or inoculated by commercial isolates and plant growth promoting rhizobacteria which may be a sustainable approach to alleviating malnutrition and supplementing human health as part of the “second green revolution” (Royal Society, 2009). Though there are many reports
∗ Corresponding author. E-mail address: [email protected] (S.C. Dutta). http://dx.doi.org/10.1016/j.scienta.2016.03.028 0304-4238/© 2016 Elsevier B.V. All rights reserved.
on AMF and plant growth promoting rhizobacteria influencing nutrient value of food crops, only a few studies have been investigated at their effects on human-relevant nutrients in edible portion of crop plants (Giovannetti et al., 2013). Recently the evaluation of antioxidant potential of food has gained much attention as antioxidants have been used as additive to prevent oxidative degradation of foods (Singh et al., 2010). Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propylgallate, and tert-butyl hydroquinone have been occupying large market shares to be added in food stuffs although they have been questioned for their carcinogenicity (Choi, 2009). Therefore, a considerable interest has been grown in the field of preventive medicine and food industry for the development of natural antioxidants. Now-a-days, food manufacturers/consumers prefer additives labeled as ‘natural’ (Gul and Bakht, 2015) and turmeric thrives a good position as a natural antioxidant (Kumar et al., 2006) having monoterpenoids, sesquiterpenoids and curcuminoids as principal phytoconstituents with antiradical properties (Ak and Gulcin, 2008; Gounder and Lingamallu, 2012). Since turmeric is extensively used as medicine and food additives, it is imperative to devise eco-friendly approaches to reduce adverse effects of agrochemicals. Turmeric plants are reported to form symbiosis with AMF (Dinesh et al., 2010; Sumathi et al., 2008; Yamawaki et al., 2013) and hence, the present study was aimed to examine on a suite of nutritional responses of turmeric plants to native isolates of AMF along with plant growth promoting
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Table 1 Soil characteristics of experimental soil collected from experimental garden of Dibrugarh University. Parameters
Quantity
Soil texture pH Moisture content (%) Organic Carbon (%) Available N (mg/kg) Available P (mg/kg)
Sandy loam 4.90–5.40 18.50–19.45 1.45–1.56 34.21–36.60 4.70–4.90
microbes. Soil fertility is one of the biggest factors responsible for the outcome of AMF inoculations on the host nutrient status (Hart et al., 2015). In North East India, agricultural soils are less fertile due to insufficient available nutrients, acidic nature of soil and minimal use of organic and inorganic fertilizers (Mahanta et al., 2012) and microbial inoculants have received increased attention for their role in management of major nutrients necessary for sustainability. Among the wide diversity of soil microbial pool, AMF and phosphate solubilizers are potent candidate for phosphorus turnover and phosphorus acquisition. Turmeric requires heavy nutrients especially nitrogen for higher yield (Hossain and Ishimine, 2007) and since nitrogen fixing bacterium Azospirillum amazonense is reported suitable for acidic soil (Magalhães et al., 1983), in our experiment a combination of nitrogen fixers, A. amazonense and Azotobacter sp. was introduced along with phosphate solubilizer Bacillus megaterium and native AMF (consortium of Glomus, Gigaspora and Acaulospora sp.) isolated from turmeric rhizosphere to facilitate growth of the turmeric plants. The objective of our study was to determine the influence of native mycorrhizal inoculation along with plant growth promoting microbes on the contents of total phenol, flavonoids and curcumin in turmeric rhizomes and antioxidant activity of methanolic extract of turmeric against DPPH and ABTS radicals. 2. Materials and methods A green house experiment was conducted from 15th March to 15th December, 2014 at the experimental farm of Dibrugarh University, Dibrugarh, Assam, located at 27◦ 5 38 N to 27◦ 42 30 N latitude and 94◦ 33 46 E to 95◦ 29 8 E longitude. The site experiences tropical monsoon climate characterized by a hot and humid atmosphere. The farm soil is sandy loam and some soil characteristics are presented in Table 1. Soil collected from the experimental garden of Dibrugarh University was sieved though a 2 mm sieve and autoclaved twice to sterilize. Turmeric rhizome fragments with at least two sprouted buds were purchased from local market of Dibrugarh Town and surface sterilized by 2.5% sodium hypochlorite solution and then washed for five to seven times with distilled water and air dried. About 20 g of rhizome fragments was planted 8 cm deep in earthen pots comprising 15 kg of sterilized soil and inoculants were applied according to different treatment types. For isolation of native AMF from turmeric rhizosphere, root, rhizome and rhizospheric soil samples (8–15 cm depth) were collected in random during spring 2012 at the time of maturity of the turmeric plants from five sampling areas of Assam i.e. Dibrugarh, Jorhat, Sonitpur, Lakhimpur and Kamrup. The AM fungal spores distributed in the soil samples were isolated in triplicate for each sample by wet sieving and decanting technique (Gerdemann and Nicolson, 1963) followed by floating-centrifugation in 50% sucrose (Dalpe, 1993). Pure culture of AMF propagules collected from turmeric fields were maintained on onion plants under sterile conditions. Identification of the spores up to genus level were done by observing the diagnostic characteristics of the spores like spore wall stratification, colour, size of the spore and type of hyphal attachment of the spore under a compound microscope
by using the manual of Schenck and Pérez (1990) and as per the description given on the INVAM website (http://invam.caf.wvu. edu/). Ten grams of soil including root bits of onion containing about 90–120 AM fungal propagules belonging to Glomus, Gigaspora and Acaulospora sp. were used as inoculants and applied along with the seed rhizome fragments. In case of control treatments equal amount of double autoclaved inoculants was added in the rhizospheric soils in the same manner. Phosphate solubilizing bacteria (B. megaterium) and nitrogen fixing microbes (Azotobacter sp. and A. amazonense) were obtained from Assam Agricultural University, Jorhat, Assam, as inorganic carrier based formulations and applied along with the seed rhizome fragments according to the manufacturer’s direction with a cfu (colony forming unit) load of 1.7 × 109 –2 × 109 ml−1 bacterial cells in different treatments. The experiment was laid out in a randomized block design and plants were irrigated on alternate days. The plants were harvested at 270 days after planting and rhizomes were cleaned of any organic debris and dried in shade. The dried rhizome of turmeric was crushed to uniform mesh and about 150 g of dried powder was extracted with solvent methanol at room temperature for 24 h. This crude extract was collected by filtering and evaporating using a rotary evaporator at 45 ◦ C. The rhizomes were examined for antioxidant activity applying both DPPH (2, 2-diphenyl-1-picrylhydrazyl radical) (Surveswaran et al., 2007) and ABTS (2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) assay (Re et al., 1999) with some modifications. The methanolic extract of turmeric rhizomes required to bring 50% of scavenging was calculated from interpolation of the curves obtained by plotting percentage inhibition against the respective concentrations of the prepared extracts. Total phenolic content (TPC) of turmeric extracts was determined by FolinCiocalteu reagent assay (Singleton et al., 1999) using gallic acid as a standard and expressed as mg gallic acid equivalents (GAE)/g dry weight. Total flavonoid content (TFC) of turmeric extracts was determined using rutin as a standard and expressed as mg rutin equivalents (RE)/g dry weight (Zhishen et al., 1999). Percentage of curcumin content was determined by ASTA method (Singh et al., 2013). For quantification of mycorrhizal colonization rate, treated turmeric roots were collected at the time of harvest and staining of the root segments was done by Trypan blue method (Phillips and Hayman, 1970) and quantified for colonization according to Giovannetti and Mosse (1980). AMF spores were also extracted from the rhizospheric soil of each treatment by wet sieving and decanting technique (Gerdemann and Nicolson, 1963) and the total number of spores was counted. AMF spore density was expressed as a number of spores per 50 g soil. P and N content of turmeric shoots were assayed by dry ashing digestion method and Kjeldahl digestion method respectively following a modification of Jones et al. (1991). The significance of treatment effects were analyzed by ANOVA using SPSS version 13. Where the F values were significant, posthoc comparison of means were evaluated by Tukey’s honestly significant difference (HSD) test at 95% confidence level (P ≤ 0.05). Regression graphs were plotted to compare the secondary metabolite contents and antioxidant activities of turmeric rhizomes and “Y” (simple linear regression) and “R2 ” (correlation) were recorded using Microsoft Excel (Version 2007).
3. Results and discussion Data pertaining to total phenolic contents (TPC), total flavonoid contents (TFC) and percentage of curcumin contents of treated plants along with IC50 values for DPPH and ABTS radical scavenging activity of tested samples was presented in Table 2. Among
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Fig. 1. Correlation between IC50 of DPPH radical scavenging activity and total phenolic contents (a), total flavonoid contents (b) and percentage of curcumin content (c) of methanolic extracts of turmeric rhizomes inoculated with native AMF consortium (Glomus, Gigaspora and Acaulospora sp.), Bacillus megaterium, Azospirillum amazonense and Azotobacter sp. Correlation between IC50 of ABTS radical scavenging activity and total phenolic contents (d), total flavonoid contents (e) and percentage of curcumin content (f) of methanolic extracts of turmeric rhizomes inoculated with native AMF consortium (Glomus, Gigaspora and Acaulospora sp.), B. megaterium, A. amazonense and Azotobacter sp.
the treatments applied, the dried rhizome of turmeric enriched with three component consortium inoculation (AMF + PSB + NF) i.e. the treatment inoculated with native AMF, Glomus, Gigaspora and
Acaulospora sp. along with phosphate solubilizer B. megaterium and nitrogen fixers A. amazonense and Azotobacter sp., showed 82.42% higher amount of phenolic content (mean: 151.54 ± 0.85 mg GAE/g)
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Table 2 Total phenolic, total flavonoid and curcumin contents and IC50 values for DPPH and ABTS radical scavenging activity of turmeric plants inoculated with native AMF consortium (Glomus, Gigaspora and Acaulospora sp.), Bacillus megaterium, Azospirillum amazonense and Azotobacter sp. Treatments
Total Phenol (mgGAE/g)
Total Flavonoids (mgRE/g)
Curcumin content (%)
IC50 for DPPH (g/ml)
IC50 for ABTS (g/ml)
Control AMF AMF + PSB AMF + NF AMF + PSB + NF
83.07 ± 0.48a 127.13 ± 0.68b 146.28 ± 0.73c 145.89 ± 1.00c 151.54 ± 0.85d
179.07 ± 0.43a 388.68 ± 1.79b 483.57 ± 1.38c 488.29 ± 1.15d 493.15 ± 1.09e
4.81 ± 0.03a 5.46 ± 0.38b 6.05 ± 0.10c 5.96 ± 0.09c 6.09 ± 0.09c
11.07 ± 0.39a 9.60 ± 0.19b 8.57 ± 0.22c 8.77 ± 0.15c 8.39 ± 0.04c
31.85 ± 0.36a 30.55 ± 0.28b 29.58 ± 0.16c 29.45 ± 0.71c 29.49 ± 0.12c
Abbreviations: AMF, consortium of Glomus, Gigaspora and Acaulospora sp.; PSB, Bacillus megaterium; NF, combination of Azospirillum amazonense and Azotobacter sp. Means with same letters in superscript at the different treatments are not significantly different at 95% confidence level (P ≤ 0.05) when compared by Tukey’s test. Treatment means are average of 5 replicates ± SD.
than uninoculated control plants (mean: 83.07 ± 0.48 mg GAE/g). Similarly, the same consortium treatment has gained 145.23% higher flavonoids (mean: 493.15 ± 1.09 mg RE/g) and 26.32% higher percentage of curcumin (mean: 6.09 ± 0.09%) over control plants (mean: 179.07 ± 0.43 mg RE/g) and (mean: 4.81 ± 0.03%) respectively. Likewise, all the tested samples showed a significant free radical scavenging activity against DPPH and ABTS radicals at IC50 range 8.39 ± 0.04 g/ml–11.07 ± 0.39 g/ml and 29.41 ± 0.71 g/ml–31.85 ± 0.36 g/ml respectively. Meanwhile, correlation analysis indicated a high correlation between the antiradical activity and the total phenolics, flavonoids and curcumin contents. Correlation coefficients of TCP, TFC, percentage of curcumin contents and anti-radical activities (DPPH and ABTS) are shown in Fig. 1. Results reveals a strong correlation with DPPH radical scavenging activity (R2 = 0.98, R2 = 0.97, R2 = 0.99) with TCP, TFP and curcumin content [Fig. 1(a–c)]. ABTS radical scavenging activity also shows a similar trend (R2 = 0.97, R2 = 0.98, R2 = 0.98) with total phenolics, total flavonoids and curcumin contents [Fig. 1(d–f)]. Table 3 represents percentage of mycorrhizal colonization in treated roots (92.60–95.80%), density of AMF spores in the turmeric rhizospheres of treated plants (285.80–309.60) per 50 g of soil and P (7.26–26.32 mg/plants) and N content of turmeric plants (191.72–319.81 mg/plants). Inoculation of turmeric plants with native AMF consortium resulted in an increase in total phenolic, flavonoids and curcumin contents in the rhizomes (Table 2). In order to determine the free radical scavenging activity of mycorrhiza inoculated turmeric rhizomes, their reactivity towards stable free radicals, DPPH and ABTS was evaluated in terms of radical reduction and expressed as IC50 (g ml−1 ). IC50 values denote the amount of tested preparations of extracts containing 50% of the contents of the inhibitor adequate to exhibit the desired activity under study. It is worthy to measure the optimal concentration of the compounds to obtain the desired function and these IC50 values might be useful in dealing of these secondary metabolites in the preparation of functional foods. The results of the present study showed that mycorrhizal treatments modulate the inhibition of both DPPH and
ABTS radical cations (Table 2) and the rise in antioxidant activities in treated plants can be explained by the presence of high phenolic and flavonoid contents particularly curcumin which is well known for its strong antioxidant activity. Phenols are aromatic secondary metabolites of plants associated with colour, sensory qualities, nutritional and antioxidant properties of edible portion of plants (Kumar et al., 2015). Flavonoids also exhibit a wide range of biochemical as well as pharmacological effects such as antioxidation, anti-inflamation, anti-platelet, anti-thrombotic action, anti-allergic etc. (Nijveldt et al., 2001). Mycorrhizal roots containing arbuscules show a fungus-induced accumulation of phynylalanine ammonia-lyase (Harrison and Dixon, 1994) which is the key enzyme for biosynthesis of phenolic compounds (Hao et al., 1996). Several researchers have reported a considerable increase in phenolic contents in tissues of host plants as a result of AM inoculation (Devi and Reddy, 2002; Cicatelli et al., 2010; Oliveira et al., 2013; Mechria et al., 2015). Besides, in our study, mycorrhizal plants exhibited 92.60–95.80% root colonization over control. A similar trend was observed with abundance of AMF spores in the rhizospheric soil of treated plants as compared to control at the time of harvest (Table 3). Mixed inoculations of AMF with other three plant growth promoting bacteria showed a significantly higher mycorrhizal colonization depicting a strong establishment of AM symbiosis. The results are in conformity with some other workers (Aseri et al., 2008) who reported a higher degree of mycorrhizal sporulation and colonization in AMF-PGPR combined inoculations as compared to mono inoculation of AMF and control. This observation could be attributed to the presence of inoculated plant growth promoting rhizobacteria which may act as a helping tool for AM establishment (Kohler et al., 2007; Aseri et al., 2008). Rhizospheric interactions imply a positive effect on soil quality, providing highly valuable ecosystem services which can be exploited in order to increase yield, reduce chemical inputs and develop an efficient form of sustainable agriculture (Owen et al., 2015). A number of previous studies have also supported increased plant growth by AMF and other phosphate solubilizing and nitrogen
Table 3 Percentage of mycorrhizal root colonization, AMF spore density in turmeric rhizosphere and P and N content of turmeric shoots inoculated with native AMF consortium (Glomus, Gigaspora and Acaulospora sp.), Bacillus megaterium, Azospirillum amazonense and Azotobacter sp. Treatments
Percentage of mycorrhizal root colonization
AMF spores per 50 g rhizospheric soil
P content of turmeric shoot (mg/plant)
N content of turmeric shoot (mg/plant)
Control AMF AMF + PSB AMF + NF AMF + PSB + NF
NDa 92.60 ± 2.60b 94.80 ± 3.56b 96.20 ± 1.30b 95.80 ± 1.09b
NDa 285.80 ± 11.58b 301.20 ± 15.89bc 309.60 ± 9.86c 301.00 ± 13.54bc
7.26 ± 0.93a 21.40 ± 0.79b 26.32 ± 0.47c 22.83 ± 1.28b 26.18 ± 0.34c
191.72 ± 6.12a 291.34 ± 5.85b 275.03 ± 3.39c 315.47 ± 6.31d 319.81 ± 10.45d
Abbreviations: AMF, consortium of Glomus, Gigaspora and Acaulospora sp.; PSB, Bacillus megaterium; NF, combination of Azospirillum amazonense and Azotobacter sp.; ND, Not Detected. Means with same letters in superscript at the different treatments are not significantly different at 95% confidence level (P ≤ 0.05) when compared by Tukey’s test. Treatment means are average of 5 replicates ± SD.
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fixing microbes (reviewed by Calvo et al., 2014; Baum et al., 2015; Corrêa et al., 2015; Pii et al., 2015). A significantly higher accumulation of secondary metabolites (total phenolics, total flavonoids and curcumin contents) in turmeric rhizome were recorded in the present study which might have resulted from enhanced plant growth and biomass production (Kohler et al., 2007; Aseri et al., 2008; Rojas-Tapias et al., 2012). AMF provide an effective mechanism to scavenge P from larger volume of soil and translocate to root cortical cells bypassing direct uptake of it by the plants, reducing the impact of inorganic P depletion in the rhizosphere (Smith et al., 2011). AMF also have been reported to directly uptake both organic and inorganic forms of nitrogen present in soil and transfer it to the host plant (Ferrol and Pérez-Tienda, 2009; Abd-Alla et al., 2014; Saia et al., 2014; Bonneau et al., 2013; Nouri et al., 2014). An enhancement in N and P uptake by the treated turmeric plants was observed in two and three component consortium inoculations of AMF with the plant growth promoting microbes over control (Table 3) which might be due to the synergistic activity of induced AMF, symbiotic nitrogen fixation by the diazotrophs (Azotobacter sp. and A. amazonense) as well as phosphate solubilization by B. megaterium. Inoculation of AMF may represent an efficient and sustainable strategy to enhance phenolic content as well as resistance to oxidative stress in plant tissues (Latef and Chaoxing, 2011; Lingua et al., 2012). Evidences indicate that AM symbiosis influences primary and secondary metabolism of host plants (Schliemann et al., 2008) inducing changes both in enzymatic activities (i.e. superoxide dismutase and catalase) (Ruiz-Lozano et al., 1996; Marin et al., 2002) and physiological mechanisms leading to the accumulation of secondary metabolites, such as carotenoids and polyphenols (Marulanda et al., 2007; Toussaint et al., 2007). The active principle of turmeric is a group of phenolic compounds including curcumin and two other curcuminoids, demethoxycurcumin and bisdemethoxycurcumin, which are well known for antioxidant activity (Singh et al., 2010). Besides, turmeric oil and oleoresin are also effective antioxidants exerting either the synergistic or additive actions towards the total antioxidant activities (Singh et al., 2010; Gounder and Lingamallu, 2012). In the present investigation, the high amount of flavonoids and phenolic contents present in all the methanolic extract of dried rhizomes might be the key factor for the observed antioxidant activity of rhizomes of turmeric (Choi, 2009; Kumar et al., 2006; Singh et al., 2010). The antioxidant activity of phenolics is mainly due to their redox properties which imparts them to react as reducing agents, hydrogen donors and singlet oxygen quenchers (Naguib et al., 2012; Skotti et al., 2014). Though numerous studies reveal strong correlation of the antioxidant activity of the plant extracts and phenolic ˇ ıcompounds present in plant tissues (Cai et al., 2006; Canadanovic´ Brunet et al., 2008; Skotti et al., 2014), some other studies have come to opposite results (Kahkonen et al., 1999) or very little correlation (Stagos et al., 2012). In our study, the positive correlation of phenolic compounds and flavonoids with anti-radical activities implies the effectiveness of the used bio-inoculants on turmeric rhizosphere.
4. Conclusion In present days, phytochemicals with functional properties have been grabbing increasing demand in food, cosmetics and pharmaceutical industries and this reinforces to study the nutritional and nutraceutical efficacy of different plant parts. In this regard, the potential of plant beneficial microbes could be an important strategy to trigger significant changes in secondary metabolites in rhizomes along with effective growth of turmeric plants despite reduction in mineral fertilizer use.
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Acknowledgement Authors are thankful to the Head, Dept. of Life Sciences, Dibrugarh University, Assam for providing the institutional support to carry out this work.
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Accumulation of secondary metabolites in response to antioxidant activity of turmeric rhizomes co-inoculated with native arbuscular mycorrhizal fungi and plant growth promoting rhizobacteria Shanti Chaya Dutta ∗ , Bijoy Neog Dept. of Life Sciences, Dibrugarh University, Dibrugarh, Assam, India
a r t i c l e
i n f o
Article history: Received 14 December 2015 Received in revised form 16 March 2016 Accepted 22 March 2016 Available online 18 April 2016 Keywords: Curcuma longa L. Bio-inoculants Antioxidant activity Flavonoids Phenolic contents Curcumin content
a b s t r a c t The present study was aimed at investigating the therapeutic potential of methanol crude extract of turmeric (Curcuma longa L.) rhizome co-inoculated with native arbuscular mycorrhizal fungi (Glomus, Gigaspora and Acaulospora sp.), phosphate solubilizer Bacillus megaterium and diazotrophic bacteria, Azospirillum amazonense and Azotobacter sp. To achieve the goals, parameters like free-radical (DPPH, ABTS) scavenging capacity, total phenolic contents, total flavonoids contents and percentage of curcumin content were examined. A significant increase of these secondary metabolites were observed in inoculated plants compared to non-inoculated controls revealing strong antioxidant activities of turmeric against DPPH and ABTS radicals in the range of 80–97%. The high amount of flavonoids (179.07–493.15 mg RE g−1 ), phenolic contents (83.07–151.54 mg GAE g−1 ) and percentage of curcumin (4.81–6.09%) present in all the methanolic extracts of dried rhizomes might be responsible for the observed antioxidant activity. © 2016 Elsevier B.V. All rights reserved.
1. Introduction A majority of human food crops are highly mycorrhizal depicting the ubiquitous nature of arbuscular mycorrhizal fungi (AMF) and the ameliorative effects of these fungi may result into some “healthier” food, either by having more concentrated nutrients (biofertilizer effect) or higher concentrations of defense-related compounds (bio-protective effect) (Antunes et al., 2012). Recently, secondary metabolites of plants which play a bioprotector role with taste and health related properties (Schreiner et al., 2012) are gaining much attention from the researchers. In this context, AMF inoculation could be an important strategy in biofortification of crops (Antunes et al., 2011). But, evidence to support their effectiveness is currently confounded by inadequate quality standards and insufficient knowledge of the underlying mechanisms, which have led to contradicting reports on field performance (Owen et al., 2015). There is, however, scope to engineer specific bioinoculant formulae like AMF, either the native ones or inoculated by commercial isolates and plant growth promoting rhizobacteria which may be a sustainable approach to alleviating malnutrition and supplementing human health as part of the “second green revolution” (Royal Society, 2009). Though there are many reports
∗ Corresponding author. E-mail address: [email protected] (S.C. Dutta). http://dx.doi.org/10.1016/j.scienta.2016.03.028 0304-4238/© 2016 Elsevier B.V. All rights reserved.
on AMF and plant growth promoting rhizobacteria influencing nutrient value of food crops, only a few studies have been investigated at their effects on human-relevant nutrients in edible portion of crop plants (Giovannetti et al., 2013). Recently the evaluation of antioxidant potential of food has gained much attention as antioxidants have been used as additive to prevent oxidative degradation of foods (Singh et al., 2010). Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propylgallate, and tert-butyl hydroquinone have been occupying large market shares to be added in food stuffs although they have been questioned for their carcinogenicity (Choi, 2009). Therefore, a considerable interest has been grown in the field of preventive medicine and food industry for the development of natural antioxidants. Now-a-days, food manufacturers/consumers prefer additives labeled as ‘natural’ (Gul and Bakht, 2015) and turmeric thrives a good position as a natural antioxidant (Kumar et al., 2006) having monoterpenoids, sesquiterpenoids and curcuminoids as principal phytoconstituents with antiradical properties (Ak and Gulcin, 2008; Gounder and Lingamallu, 2012). Since turmeric is extensively used as medicine and food additives, it is imperative to devise eco-friendly approaches to reduce adverse effects of agrochemicals. Turmeric plants are reported to form symbiosis with AMF (Dinesh et al., 2010; Sumathi et al., 2008; Yamawaki et al., 2013) and hence, the present study was aimed to examine on a suite of nutritional responses of turmeric plants to native isolates of AMF along with plant growth promoting
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Table 1 Soil characteristics of experimental soil collected from experimental garden of Dibrugarh University. Parameters
Quantity
Soil texture pH Moisture content (%) Organic Carbon (%) Available N (mg/kg) Available P (mg/kg)
Sandy loam 4.90–5.40 18.50–19.45 1.45–1.56 34.21–36.60 4.70–4.90
microbes. Soil fertility is one of the biggest factors responsible for the outcome of AMF inoculations on the host nutrient status (Hart et al., 2015). In North East India, agricultural soils are less fertile due to insufficient available nutrients, acidic nature of soil and minimal use of organic and inorganic fertilizers (Mahanta et al., 2012) and microbial inoculants have received increased attention for their role in management of major nutrients necessary for sustainability. Among the wide diversity of soil microbial pool, AMF and phosphate solubilizers are potent candidate for phosphorus turnover and phosphorus acquisition. Turmeric requires heavy nutrients especially nitrogen for higher yield (Hossain and Ishimine, 2007) and since nitrogen fixing bacterium Azospirillum amazonense is reported suitable for acidic soil (Magalhães et al., 1983), in our experiment a combination of nitrogen fixers, A. amazonense and Azotobacter sp. was introduced along with phosphate solubilizer Bacillus megaterium and native AMF (consortium of Glomus, Gigaspora and Acaulospora sp.) isolated from turmeric rhizosphere to facilitate growth of the turmeric plants. The objective of our study was to determine the influence of native mycorrhizal inoculation along with plant growth promoting microbes on the contents of total phenol, flavonoids and curcumin in turmeric rhizomes and antioxidant activity of methanolic extract of turmeric against DPPH and ABTS radicals. 2. Materials and methods A green house experiment was conducted from 15th March to 15th December, 2014 at the experimental farm of Dibrugarh University, Dibrugarh, Assam, located at 27◦ 5 38 N to 27◦ 42 30 N latitude and 94◦ 33 46 E to 95◦ 29 8 E longitude. The site experiences tropical monsoon climate characterized by a hot and humid atmosphere. The farm soil is sandy loam and some soil characteristics are presented in Table 1. Soil collected from the experimental garden of Dibrugarh University was sieved though a 2 mm sieve and autoclaved twice to sterilize. Turmeric rhizome fragments with at least two sprouted buds were purchased from local market of Dibrugarh Town and surface sterilized by 2.5% sodium hypochlorite solution and then washed for five to seven times with distilled water and air dried. About 20 g of rhizome fragments was planted 8 cm deep in earthen pots comprising 15 kg of sterilized soil and inoculants were applied according to different treatment types. For isolation of native AMF from turmeric rhizosphere, root, rhizome and rhizospheric soil samples (8–15 cm depth) were collected in random during spring 2012 at the time of maturity of the turmeric plants from five sampling areas of Assam i.e. Dibrugarh, Jorhat, Sonitpur, Lakhimpur and Kamrup. The AM fungal spores distributed in the soil samples were isolated in triplicate for each sample by wet sieving and decanting technique (Gerdemann and Nicolson, 1963) followed by floating-centrifugation in 50% sucrose (Dalpe, 1993). Pure culture of AMF propagules collected from turmeric fields were maintained on onion plants under sterile conditions. Identification of the spores up to genus level were done by observing the diagnostic characteristics of the spores like spore wall stratification, colour, size of the spore and type of hyphal attachment of the spore under a compound microscope
by using the manual of Schenck and Pérez (1990) and as per the description given on the INVAM website (http://invam.caf.wvu. edu/). Ten grams of soil including root bits of onion containing about 90–120 AM fungal propagules belonging to Glomus, Gigaspora and Acaulospora sp. were used as inoculants and applied along with the seed rhizome fragments. In case of control treatments equal amount of double autoclaved inoculants was added in the rhizospheric soils in the same manner. Phosphate solubilizing bacteria (B. megaterium) and nitrogen fixing microbes (Azotobacter sp. and A. amazonense) were obtained from Assam Agricultural University, Jorhat, Assam, as inorganic carrier based formulations and applied along with the seed rhizome fragments according to the manufacturer’s direction with a cfu (colony forming unit) load of 1.7 × 109 –2 × 109 ml−1 bacterial cells in different treatments. The experiment was laid out in a randomized block design and plants were irrigated on alternate days. The plants were harvested at 270 days after planting and rhizomes were cleaned of any organic debris and dried in shade. The dried rhizome of turmeric was crushed to uniform mesh and about 150 g of dried powder was extracted with solvent methanol at room temperature for 24 h. This crude extract was collected by filtering and evaporating using a rotary evaporator at 45 ◦ C. The rhizomes were examined for antioxidant activity applying both DPPH (2, 2-diphenyl-1-picrylhydrazyl radical) (Surveswaran et al., 2007) and ABTS (2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) assay (Re et al., 1999) with some modifications. The methanolic extract of turmeric rhizomes required to bring 50% of scavenging was calculated from interpolation of the curves obtained by plotting percentage inhibition against the respective concentrations of the prepared extracts. Total phenolic content (TPC) of turmeric extracts was determined by FolinCiocalteu reagent assay (Singleton et al., 1999) using gallic acid as a standard and expressed as mg gallic acid equivalents (GAE)/g dry weight. Total flavonoid content (TFC) of turmeric extracts was determined using rutin as a standard and expressed as mg rutin equivalents (RE)/g dry weight (Zhishen et al., 1999). Percentage of curcumin content was determined by ASTA method (Singh et al., 2013). For quantification of mycorrhizal colonization rate, treated turmeric roots were collected at the time of harvest and staining of the root segments was done by Trypan blue method (Phillips and Hayman, 1970) and quantified for colonization according to Giovannetti and Mosse (1980). AMF spores were also extracted from the rhizospheric soil of each treatment by wet sieving and decanting technique (Gerdemann and Nicolson, 1963) and the total number of spores was counted. AMF spore density was expressed as a number of spores per 50 g soil. P and N content of turmeric shoots were assayed by dry ashing digestion method and Kjeldahl digestion method respectively following a modification of Jones et al. (1991). The significance of treatment effects were analyzed by ANOVA using SPSS version 13. Where the F values were significant, posthoc comparison of means were evaluated by Tukey’s honestly significant difference (HSD) test at 95% confidence level (P ≤ 0.05). Regression graphs were plotted to compare the secondary metabolite contents and antioxidant activities of turmeric rhizomes and “Y” (simple linear regression) and “R2 ” (correlation) were recorded using Microsoft Excel (Version 2007).
3. Results and discussion Data pertaining to total phenolic contents (TPC), total flavonoid contents (TFC) and percentage of curcumin contents of treated plants along with IC50 values for DPPH and ABTS radical scavenging activity of tested samples was presented in Table 2. Among
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Fig. 1. Correlation between IC50 of DPPH radical scavenging activity and total phenolic contents (a), total flavonoid contents (b) and percentage of curcumin content (c) of methanolic extracts of turmeric rhizomes inoculated with native AMF consortium (Glomus, Gigaspora and Acaulospora sp.), Bacillus megaterium, Azospirillum amazonense and Azotobacter sp. Correlation between IC50 of ABTS radical scavenging activity and total phenolic contents (d), total flavonoid contents (e) and percentage of curcumin content (f) of methanolic extracts of turmeric rhizomes inoculated with native AMF consortium (Glomus, Gigaspora and Acaulospora sp.), B. megaterium, A. amazonense and Azotobacter sp.
the treatments applied, the dried rhizome of turmeric enriched with three component consortium inoculation (AMF + PSB + NF) i.e. the treatment inoculated with native AMF, Glomus, Gigaspora and
Acaulospora sp. along with phosphate solubilizer B. megaterium and nitrogen fixers A. amazonense and Azotobacter sp., showed 82.42% higher amount of phenolic content (mean: 151.54 ± 0.85 mg GAE/g)
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Table 2 Total phenolic, total flavonoid and curcumin contents and IC50 values for DPPH and ABTS radical scavenging activity of turmeric plants inoculated with native AMF consortium (Glomus, Gigaspora and Acaulospora sp.), Bacillus megaterium, Azospirillum amazonense and Azotobacter sp. Treatments
Total Phenol (mgGAE/g)
Total Flavonoids (mgRE/g)
Curcumin content (%)
IC50 for DPPH (g/ml)
IC50 for ABTS (g/ml)
Control AMF AMF + PSB AMF + NF AMF + PSB + NF
83.07 ± 0.48a 127.13 ± 0.68b 146.28 ± 0.73c 145.89 ± 1.00c 151.54 ± 0.85d
179.07 ± 0.43a 388.68 ± 1.79b 483.57 ± 1.38c 488.29 ± 1.15d 493.15 ± 1.09e
4.81 ± 0.03a 5.46 ± 0.38b 6.05 ± 0.10c 5.96 ± 0.09c 6.09 ± 0.09c
11.07 ± 0.39a 9.60 ± 0.19b 8.57 ± 0.22c 8.77 ± 0.15c 8.39 ± 0.04c
31.85 ± 0.36a 30.55 ± 0.28b 29.58 ± 0.16c 29.45 ± 0.71c 29.49 ± 0.12c
Abbreviations: AMF, consortium of Glomus, Gigaspora and Acaulospora sp.; PSB, Bacillus megaterium; NF, combination of Azospirillum amazonense and Azotobacter sp. Means with same letters in superscript at the different treatments are not significantly different at 95% confidence level (P ≤ 0.05) when compared by Tukey’s test. Treatment means are average of 5 replicates ± SD.
than uninoculated control plants (mean: 83.07 ± 0.48 mg GAE/g). Similarly, the same consortium treatment has gained 145.23% higher flavonoids (mean: 493.15 ± 1.09 mg RE/g) and 26.32% higher percentage of curcumin (mean: 6.09 ± 0.09%) over control plants (mean: 179.07 ± 0.43 mg RE/g) and (mean: 4.81 ± 0.03%) respectively. Likewise, all the tested samples showed a significant free radical scavenging activity against DPPH and ABTS radicals at IC50 range 8.39 ± 0.04 g/ml–11.07 ± 0.39 g/ml and 29.41 ± 0.71 g/ml–31.85 ± 0.36 g/ml respectively. Meanwhile, correlation analysis indicated a high correlation between the antiradical activity and the total phenolics, flavonoids and curcumin contents. Correlation coefficients of TCP, TFC, percentage of curcumin contents and anti-radical activities (DPPH and ABTS) are shown in Fig. 1. Results reveals a strong correlation with DPPH radical scavenging activity (R2 = 0.98, R2 = 0.97, R2 = 0.99) with TCP, TFP and curcumin content [Fig. 1(a–c)]. ABTS radical scavenging activity also shows a similar trend (R2 = 0.97, R2 = 0.98, R2 = 0.98) with total phenolics, total flavonoids and curcumin contents [Fig. 1(d–f)]. Table 3 represents percentage of mycorrhizal colonization in treated roots (92.60–95.80%), density of AMF spores in the turmeric rhizospheres of treated plants (285.80–309.60) per 50 g of soil and P (7.26–26.32 mg/plants) and N content of turmeric plants (191.72–319.81 mg/plants). Inoculation of turmeric plants with native AMF consortium resulted in an increase in total phenolic, flavonoids and curcumin contents in the rhizomes (Table 2). In order to determine the free radical scavenging activity of mycorrhiza inoculated turmeric rhizomes, their reactivity towards stable free radicals, DPPH and ABTS was evaluated in terms of radical reduction and expressed as IC50 (g ml−1 ). IC50 values denote the amount of tested preparations of extracts containing 50% of the contents of the inhibitor adequate to exhibit the desired activity under study. It is worthy to measure the optimal concentration of the compounds to obtain the desired function and these IC50 values might be useful in dealing of these secondary metabolites in the preparation of functional foods. The results of the present study showed that mycorrhizal treatments modulate the inhibition of both DPPH and
ABTS radical cations (Table 2) and the rise in antioxidant activities in treated plants can be explained by the presence of high phenolic and flavonoid contents particularly curcumin which is well known for its strong antioxidant activity. Phenols are aromatic secondary metabolites of plants associated with colour, sensory qualities, nutritional and antioxidant properties of edible portion of plants (Kumar et al., 2015). Flavonoids also exhibit a wide range of biochemical as well as pharmacological effects such as antioxidation, anti-inflamation, anti-platelet, anti-thrombotic action, anti-allergic etc. (Nijveldt et al., 2001). Mycorrhizal roots containing arbuscules show a fungus-induced accumulation of phynylalanine ammonia-lyase (Harrison and Dixon, 1994) which is the key enzyme for biosynthesis of phenolic compounds (Hao et al., 1996). Several researchers have reported a considerable increase in phenolic contents in tissues of host plants as a result of AM inoculation (Devi and Reddy, 2002; Cicatelli et al., 2010; Oliveira et al., 2013; Mechria et al., 2015). Besides, in our study, mycorrhizal plants exhibited 92.60–95.80% root colonization over control. A similar trend was observed with abundance of AMF spores in the rhizospheric soil of treated plants as compared to control at the time of harvest (Table 3). Mixed inoculations of AMF with other three plant growth promoting bacteria showed a significantly higher mycorrhizal colonization depicting a strong establishment of AM symbiosis. The results are in conformity with some other workers (Aseri et al., 2008) who reported a higher degree of mycorrhizal sporulation and colonization in AMF-PGPR combined inoculations as compared to mono inoculation of AMF and control. This observation could be attributed to the presence of inoculated plant growth promoting rhizobacteria which may act as a helping tool for AM establishment (Kohler et al., 2007; Aseri et al., 2008). Rhizospheric interactions imply a positive effect on soil quality, providing highly valuable ecosystem services which can be exploited in order to increase yield, reduce chemical inputs and develop an efficient form of sustainable agriculture (Owen et al., 2015). A number of previous studies have also supported increased plant growth by AMF and other phosphate solubilizing and nitrogen
Table 3 Percentage of mycorrhizal root colonization, AMF spore density in turmeric rhizosphere and P and N content of turmeric shoots inoculated with native AMF consortium (Glomus, Gigaspora and Acaulospora sp.), Bacillus megaterium, Azospirillum amazonense and Azotobacter sp. Treatments
Percentage of mycorrhizal root colonization
AMF spores per 50 g rhizospheric soil
P content of turmeric shoot (mg/plant)
N content of turmeric shoot (mg/plant)
Control AMF AMF + PSB AMF + NF AMF + PSB + NF
NDa 92.60 ± 2.60b 94.80 ± 3.56b 96.20 ± 1.30b 95.80 ± 1.09b
NDa 285.80 ± 11.58b 301.20 ± 15.89bc 309.60 ± 9.86c 301.00 ± 13.54bc
7.26 ± 0.93a 21.40 ± 0.79b 26.32 ± 0.47c 22.83 ± 1.28b 26.18 ± 0.34c
191.72 ± 6.12a 291.34 ± 5.85b 275.03 ± 3.39c 315.47 ± 6.31d 319.81 ± 10.45d
Abbreviations: AMF, consortium of Glomus, Gigaspora and Acaulospora sp.; PSB, Bacillus megaterium; NF, combination of Azospirillum amazonense and Azotobacter sp.; ND, Not Detected. Means with same letters in superscript at the different treatments are not significantly different at 95% confidence level (P ≤ 0.05) when compared by Tukey’s test. Treatment means are average of 5 replicates ± SD.
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fixing microbes (reviewed by Calvo et al., 2014; Baum et al., 2015; Corrêa et al., 2015; Pii et al., 2015). A significantly higher accumulation of secondary metabolites (total phenolics, total flavonoids and curcumin contents) in turmeric rhizome were recorded in the present study which might have resulted from enhanced plant growth and biomass production (Kohler et al., 2007; Aseri et al., 2008; Rojas-Tapias et al., 2012). AMF provide an effective mechanism to scavenge P from larger volume of soil and translocate to root cortical cells bypassing direct uptake of it by the plants, reducing the impact of inorganic P depletion in the rhizosphere (Smith et al., 2011). AMF also have been reported to directly uptake both organic and inorganic forms of nitrogen present in soil and transfer it to the host plant (Ferrol and Pérez-Tienda, 2009; Abd-Alla et al., 2014; Saia et al., 2014; Bonneau et al., 2013; Nouri et al., 2014). An enhancement in N and P uptake by the treated turmeric plants was observed in two and three component consortium inoculations of AMF with the plant growth promoting microbes over control (Table 3) which might be due to the synergistic activity of induced AMF, symbiotic nitrogen fixation by the diazotrophs (Azotobacter sp. and A. amazonense) as well as phosphate solubilization by B. megaterium. Inoculation of AMF may represent an efficient and sustainable strategy to enhance phenolic content as well as resistance to oxidative stress in plant tissues (Latef and Chaoxing, 2011; Lingua et al., 2012). Evidences indicate that AM symbiosis influences primary and secondary metabolism of host plants (Schliemann et al., 2008) inducing changes both in enzymatic activities (i.e. superoxide dismutase and catalase) (Ruiz-Lozano et al., 1996; Marin et al., 2002) and physiological mechanisms leading to the accumulation of secondary metabolites, such as carotenoids and polyphenols (Marulanda et al., 2007; Toussaint et al., 2007). The active principle of turmeric is a group of phenolic compounds including curcumin and two other curcuminoids, demethoxycurcumin and bisdemethoxycurcumin, which are well known for antioxidant activity (Singh et al., 2010). Besides, turmeric oil and oleoresin are also effective antioxidants exerting either the synergistic or additive actions towards the total antioxidant activities (Singh et al., 2010; Gounder and Lingamallu, 2012). In the present investigation, the high amount of flavonoids and phenolic contents present in all the methanolic extract of dried rhizomes might be the key factor for the observed antioxidant activity of rhizomes of turmeric (Choi, 2009; Kumar et al., 2006; Singh et al., 2010). The antioxidant activity of phenolics is mainly due to their redox properties which imparts them to react as reducing agents, hydrogen donors and singlet oxygen quenchers (Naguib et al., 2012; Skotti et al., 2014). Though numerous studies reveal strong correlation of the antioxidant activity of the plant extracts and phenolic ˇ ıcompounds present in plant tissues (Cai et al., 2006; Canadanovic´ Brunet et al., 2008; Skotti et al., 2014), some other studies have come to opposite results (Kahkonen et al., 1999) or very little correlation (Stagos et al., 2012). In our study, the positive correlation of phenolic compounds and flavonoids with anti-radical activities implies the effectiveness of the used bio-inoculants on turmeric rhizosphere.
4. Conclusion In present days, phytochemicals with functional properties have been grabbing increasing demand in food, cosmetics and pharmaceutical industries and this reinforces to study the nutritional and nutraceutical efficacy of different plant parts. In this regard, the potential of plant beneficial microbes could be an important strategy to trigger significant changes in secondary metabolites in rhizomes along with effective growth of turmeric plants despite reduction in mineral fertilizer use.
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Acknowledgement Authors are thankful to the Head, Dept. of Life Sciences, Dibrugarh University, Assam for providing the institutional support to carry out this work.
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