Effect of induced stress treatments on Zingiber officinale rosc. Cv-Varada with respect to its growth dynamics and phytochemical characterization

hytochemicals are bio- active chemicals of plant origin. They are regarded as secondary metabolites because the plant that manufactures them may have little need for them. They are naturally synthesized in all parts of the plant body; bark, leaves stem, root, flower, fruits, seeds, etc. i.e. any part of the plant body may contain active components (Tiwari et al., 2011). Natural antioxidants or phytochemical are secondary metabolites of plants namely carotenoids, flavonoids, cinnamic acids, benzoic acids, folic acid, ascorbic acid, tocopherols, tocotrienols, etc. are among the antioxidants produced by plants for their own sustenance. Beta-carotene, ascorbic acid and alpha tocopherols are widely used antioxidants. Zingiber officinale contains a number of antioxidants such as beta-carotene, ascorbic acid, terpenoids, alkaloids, and polyphenols such as flavonoids, flavones glycosides, rutin, etc. (Ghasemzadeh, 2010).

Z. officinale is one of the most widely used herbs and food flavouring agent.Zingiber comprising of 150 species and four sections distributed throughout tropical Asia, China, Japan and tropical Australia besides the subspecies (varieties): Z. officinale var. rubra and Z. officinale var. rubrum (Muda et al., 2004). Z. officinale is known by different names in different parts of world. It is called different names in different part of the world e.g. Zenzero in Italian, Jeung or Sang Keong in Chinese, Aliah in Indonesia, Adrack in Urdu, Gember in Dutch, Jengibre in Spanish, Ingwar in German, Gingembre in French (Kumar et al., 2011).

In medicinal and aromatic plants, growth and biosynthesis of secondary metabolites are strongly influenced by genetic, environmental factors, and genetic×environmental effects ( irbalouti et al., 2014). lant physiologists have been looking for new alternatives to conventional methods for improvimg in production of secondary metabolites. One of these methods is elicitation, which it can be an important strategy towards obtaining improved production of bioactive compounds (Bajalan et al., 2017). Some chemical compounds that could be used as elicitors to modify secondary metabolites and subsequentlythe bioactivity of medicinal and aromatic plants. Recent years, the applications of signal components as elicitors have evolved an effective strategy for the production of target secondary metabolites in plant ( irbalouti et al., 2014).

MATERIALS AND METHODS

The study was conducted in rain-protected green house maintained. Zingiber officinale cv- varada was used for the present study. Ginger seed rhizomes were collected from Indian Institute of Spices Research, Kozhikode (ICAR Unit- IISR Kozhikode) and maintained in the greenhouse of Department of Botany, St. Joseph’s College, Devagiri, Kozhikode, Kerala at 28⁰C and the experimental area lies between 11.2588^o N, 75.7804^o E. Rhizomes were grown in grow bags containing soil and coco peat in the ratio 2:1. p^H of the soil 5.66, electrical conductivity 0.0302 S/m. There were about 15 grow bags kept for every single stress treatment.

Stress Treatments

When the ginger seedlings were at the second leaf stage, (approximately 90 days after planting) they were sprayed with two concentrations (10-2 and 10-3 M) of salicylic acid solution (SA; 2-hydroxybenzoic acid + 100 μl dimethyl sulfoxide + 0.02% olyoxyethylenesorbitan monolaurate, Tween 20, Sigma Chemicals; pH 6.5), Zinc sulphate with same molar concentrations (ZnSO4 7 H2O dissolved in distilled water +   0.02% olyoxyethylenesorbitan monolaurate, Tween 20, Sigma Chemicals; pH 4). Control plants were sprayed with same solution but without SA and ZnSO4. A trial was also maintained without any foliar application to estimate the amount of non volatile components of the variety under study. Apart from foliar spray drought stress is imposed by withholding water application, at the trials. lants were sprayed once on the leaves early in the morning and every week until one month.

Morphological Analysis

Five plants/ treatments / replication were collected at random and studied for growth characters. erformance of ginger varieties for growth attributes like plant height, numbers of leaves per plant, number of tillers per plant were evaluated after 180 days of planting and yield attributes like weight of fresh rhizome were calculated at the end of harvest and the data was recorded.

Preparation of Extracts

Five grams of the dry powder from rhizome were weighed carefully and done hot methanolic extraction. The extracts were kept for 72 hours and then it is made up to 50ml with methanol.

Phytochemical AnalysisEstimation of antioxidant activity (DPPH Assay)

The antioxidant capacity of the extracts from ginger under various treatments was evaluated by D H assay (Hung et al., 2005). Decreasing of D H solution absorbance indicates an increase of D H radical scavenging activity. The amount of sample necessary to decrease the absorbance of D H (Sigma–Aldrich Co., Steineheim, Germany)

Estimation of total phenolics

The total phenolic content in the extracts was determined by theFolin–Ciocalteu (Sigma–Aldrich Co., Steineheim, Germany) assay (Singleton and Rossi, 1965). The absorbance of the samples was measured at 650 nm against a reagent blank using a UV–vis spectrophotometer (Thermoscientific Genesys 50). Gallic acid (Merck Co., Darmstadt, Germany) equivalent (GAL) was used as the reference.

Estimation of total flavonoids

Total flavonoids content in the extracts from sage under various treatments was determined according. Ordonez et al. , (2006) based on the formation of a flavonoid–aluminum complex with a maximum absorptivity at 510 nm. The flavonoids content is expressed as mg quercetin (Roth Co., Karlsruhe,

Germany) equivalents per gram of each extract on dry basis mg quercetin/100g dry weight.

RESULTS AND DISCUSSION

Single variety of ginger ( Zingiber officinale cv- varada ) under various solitary as well as combined stress treatments were evaluated for variation in morphological as well as in preliminary phytochemical analysis. Morphological parameters under study include lant height, Number of leaves per plant, Number of tillers per plant and yield attributes characterized by fresh and dry weight of the rhizome.

A study on Thymus vulgaris by (Khalil et al., 2018) showed that decreasing field capacity and increasing SA concentration had a significant effect on height and fresh and dry weights of the plant compared to control plants. It was observed that foliar application of SA reversed the negative impact of drought stress on these three parameters at up to 2mM concentration.

Total Phenolics and Flavonoids

henolic compounds contribute directly for antioxidative action and they constitute the main class of natural antioxidants present in the herbs (Awika et al., 2003), therefore it is necessary to calculate amounts of phenolic compounds under different conditions. From the results it can be inferred that increasing concentration of foliar spray of salicylic acid has a positive impact on the total phenolic content when comparing with the foliar application zinc sulphate of same concentration.

Results of the present study showed significant differences in contents of total phenolic and flavonoid of the methanolic extract of ginger powder under different treatments (Table 3). The highest amounts of total phenolic of the extracts were obtained from combined stress treatments. Similarly, apart from solitary application of zinc sulphate and salicyilic acid combined with drought enhanced the total phenolics content. Bistgani et al,. (2017a) reported that mild drought stress caused an increase in amount of phenolic compounds in T. daenensis . In addition, results a study by Manukyan (2011) indicated that drought stress affected positively polyphenolic content in Melissa officinalis L. In general, water deficit increases polyphenolic content of more herbs, because in case of stress, more metabolites are produced in the plants and substances prevent from oxidization in the cells. On the other hand, some researchers have observed quite the opposite effect. For example, Bistgani et al., (2017b) reported that drought stress inhibits the total synthesis of flavonoid in T. daenensis leaves and shoots.

The same trend is observed in the case of phenolics is followed here also. Combination of stresses resulted in higher flavonoid content. Here also foliar spray of salicylic acid shows a greater impact on the concentration of flavonoids than that of the foliar application of zinc sulphate. The study by Hassanpouraghdam, et al., (2019) showed that, there exist a positive effect on flavonoids by foliar spray zinc and iron on Rosmarinus officinalis. Combined effect of CO 2 enrichment and foliar application of salicylic acid on the production and antioxidant activities of anthocyanin, flavonoids and isoflavonoids from ginger was studied by Ghasemzadeh et al., (2012).

Antioxidant Assay by Dpph

The D H free radical scavenging activity is one of those indicators which are important in determining antioxidant potential of selected bioactive molecules or extract containing them. Antioxidant activities of the extract were evaluated using D H and represented in the table. Results of this study for free radical scavenging activity are statistically significantly different among different treatments (Table 6). The highest antioxidant activity of the extracts of sage was obtained from reduced irrigation with foliar spray of salicylic acid at high concentration treatment by D H assay (Table 6). These results can be attributed to the higher levels of polyphenolic (total phenol and flavonoid) in comparison to the well-watered plants. The results suggested that the phenolic components contributed significantly to the antioxidant capacity of the medicinal and aromatic plants ( irbalouti et al., 2014). Several investigations ( roteggente et al., 2002; Corral-Aguayo et al., 2008; Bajalan et al., 2017) have studied correlations between bioactive compounds and antioxidant activity by D H assay in numerous herbs and spices. Literature review shows the presence of the two main groups of secondary metabolites in the genus of Salvia, volatile terpenoids and polyphenolic compounds (Roby et al., 2013; Martins et al., 2015) which are mainly responsible for the biological effects of the genus, particularly antioxidant activities. Interaction effects of foliar spray of salicylic acid×irrigation frequencies significantly influenced on the antioxidant activities by D H assay of the extracts (Table 4). The highest antioxidant activities in plants sprayed with 10 -2 Molar salicylic acid under reduced irrigation condition (Table 6). robably, Salicylic acid could regulate the activities of antioxidant enzymes and increase plant tolerance to abiotic stresses. henolic compounds are believed to account for a major portion of the antioxidant capacity in many plants (Yogesh et al., 2012b). Epidemio-logical studies suggest that the consumption of flavonoid-rich foods protects against human diseases associated with oxidative stress. In vitro, flavonoids from several plant sources have shown free-radicalscavenging activity and protection against oxidative stress ( irbalouti et al., 2014b). At the same time foliar spray of zinc sulphate also had positive effect on the antioxidant activity. From the table it can also be inferred that the combination of stresses had a major impact on all the properties under study. Thus this crosstalk between different stresses is main reason for the increased amount of phenolics, flavonoids and antioxidant under study.

DPPH Assay

The radical scavenging effect of 15 samples were calculated and listed in table 7.

Table-1 erformance of ginger variety ( Zingiber officinale cv-varada) for growth attributes (After 180 days of planting)

Treatments	Height of the plant(cm)	No.of leaves per plant	No.of tillers per plant
SA 10-2molar	90.2	14.33	3.5
SA 10-3 molar	84.2	14.1	2
SA 10-2molar+ drought	86.3	13	2
SA 10-3 molar+ drought	85	13.21	1
ZnSo 4 (C)	72.8	10.7	2
ZnSo 4 10-2molar	73.7	10.8	1
ZnSo 4 10-3 molar	76.8	11	1
ZnSo 4 10-2Molar+ drought	77	12	1
ZnSo 4 10-3 molar+ drought	78	13.5	2
SA 10-3 molar +ZnSo 4 10-3 molar	75	12.8	1
SA 10-2molar +ZnSo 4 10-2molar	85	13.5	1
SA 10-3 molar+ ZnSo 4 10-2 molar	84	12.9	1
	82	11.2	1

Table-2 erformance of ginger variety ( Zingiber officinale cv-varada) for yield attributes (After 180 days of planting)

Treatments	Rhizome fresh weight (in g)	Rhizome dry weight (in g)
Control	275	44.15
SA 10-2molar	435	60.484
SA 10-3 molar	400	55.9
SA 10-2molar+ drought	305	51
SA 10-3 molar+ drought	280	48.7
ZnSO 4 10-2molar	355	52
ZnSO 4 10-3 molar	275	47
ZnSO 4 10-2molar+ drought	210	40.4
ZnSO 4 10-3 molar+ drought	270	42.1
SA 10-3 molar +ZnSO 4 10-3 molar	245	43
SA 10-2molar +ZnSO 4 10-2molar	260	47
SA 10-3 molar+ ZnSO 4 10-2 molar	256	45.2
SA 10-2 molar+ ZnSO 4 10-3 molar	258	45

Table 3 The absorbance of different concentration of GA at 650nm

Concentration of quercetin ( μg/ml)	Absorbance at 510 nm
200	0.161
400 600 800 1000	0.366 0.489 0.559 0.820

Table-4 The absorbance of different samples and its corresponding concentration of total phenolics obtained from the standard graph

Treatments	Absorbance ( at 6 0nm)	GA equivalent (mg GA/g)
Control	0.732	13.2881
SA 10-2molar	1.7865	34.4605
SA 10-3 molar	1.0325	19.3214
SA 10-2molar+ drought	1.2315	23.31692
SA 10-3 molar+ drought	1.2275	23.23661
ZnSO 4 10-2molar	0.8915	16.4905
ZnSO 4 10-3 molar	0.761	13.87035
ZnSO 4 10-2molar+ drought	1.3905	26.50928
ZnSO 4 10-3 molar+ drought	1.469	28.08539
SA 10-3 molar +ZnSO 4 10-3 molar	1.8005	34.74115
SA 10-2molar +ZnSO 4 10-2molar	2.268	44.12749
SA 10-3 molar+ ZnSO 4 10-2 molar	1.9585	37.9143
SA 10-2 molar+ ZnSO 4 10-3 molar	2.089	40.5335

Table- showing the absorbance of different concentration of Quercetin at 10nm

Concentration of GA ( μg/ml)	Absorbance at 650nm
5	0.228
10	0.685
20	1.099
30	1.498
40	2.10
50	2.531

Table-6 Showing the absorbance of different samples and its corresponding concentration of total Flavonoids obtained from the standard graph

Treatments	Absorbance ( at 10nm)	Quercetin equivalent (mg Q/g)
Control	0.64	807.9709
SA 10-2molar	0.74	933.25
SA 10-3 molar	0.66	833.027
SA 10-2molar+ drought	0.78	983.368
SA 10-3 molar+ drought	0.75	945.78
ZnSO 4 10-2molar	0.64	807.9709
ZnSO 4 10-3 molar	0.52	657.6
ZnSO 4 10-2molar+ drought	0.69	870.6127
ZnSO 4 10-3 molar+ drought	0.65	820.4992
SA 10-3 molar +ZnSO 4 10-3 molar	0.692	873.1183
SA 10-2molar +ZnSO 4 10-2molar	0.77	970.83
SA 10-3 molar+ ZnSO 4 10-2 molar	0.69	870.6127
SA 10-2 molar+ ZnSO 4 10-3 molar	0.65	820.4992

Table-7 Showing the absorbance of different samples and its corresponding Percentage inhibition calculated at 17 nm

Treatments	Absorbance ( at 6 0nm)	GA equivalent (mg GA/g)
Control	0.732	13.2881
SA 10-2molar	1.7865	34.4605
SA 10-3 molar	1.0325	19.3214
SA 10-2molar+ drought	1.2315	23.31692
SA 10-3 molar+ drought	1.2275	23.23661
ZnSO 4 10-2molar	0.8915	16.4905
ZnSO 4 10-3 molar	0.761	13.87035
ZnSO 4 10-2molar+ drought	1.3905	26.50928
ZnSO 4 10-3 molar+ drought	1.469	28.08539
SA 10-3 molar +ZnSO 4 10-3 molar	1.8005	34.74115
SA 10-2molar +ZnSO 4 10-2molar	2.268	44.12749
SA 10-3 molar+ ZnSO 4 10-2 molar	1.9585	37.9143
SA 10-2 molar+ ZnSO 4 10-3 molar	2.089	40.5335

Figure 1. GA standard Graph for henolics

Figure 2. Quercetin standard graph for Flavonoids

CONCLUSIONS

This investigation demonstrated that stressful conditions stimulated the synthesis of secondary metabolites. Aerial spraying with SA and ZnSO 4 compensated to some degree for the negative impacts of reduced irrigation on production of some secondary metabolites total phenolic and flavonoid in the extract. The trials which shown maximum antioxidant activity are those with high amount of total phenolics. From these results, it is believed that SA is modifying the activity of enzymes responsible for the bioformation of ginger phenolics. In this sense phenyl-alanine lyase, which is the first enzyme regulating the phenylpropanoid pathway has been already reported to be affected by phytoregulators. As a result, the effect of SA on phenylalanine lyase activity would eventually result in an alteration of the bioformation of phenolics.

From the given study it could also be inferred that there occurs a crosstalk between the stress signals which could be the possible reason for the difference in the total phenolics, total flavonoids and antioxidant activity. From the results it can be inferred that increasing concentration of foliar spray of salicylic acid has a positive impact on the total phenolic content when comparing with the foliar application zinc sulphate of same concentration.

The present study suggests that Z. officinale for further research on improving the quality and phytochemical value of ginger by using various elicitors. This also provides a greater opportunity in studying and analyzing the molecular mechanism and the signal transduction pathway and how one signal interfere (could be both positively or negatively) with the transduction pathway or another signal. After all, Ginger is one of the major spice, for which drought is considered as a major menace, can be effectively overcome by this cross talk between different stress signals.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the funding agency, the University Grant Commission (UGC) of the Government of India, (Grant number 366088) for providing financial support, in the form of UGC-CSIR NET-JRF/SRF to the First author.

CONFLICTS OF INTEREST

The authors declare that they have no potential conflicts of interest.