Introduction

Ruta graveolens L. (rue), an evergreen herb or a small shrub belonging to the family Rutaceae is a well explored medicinal plant. It is beneficial for the treatment of neuromuscular problems, gastric disorders, stiff neck, dizziness, rheumatism, eyestrain-induced headache and inflammation (Raghav et al. 2006). These therapeutic properties are attributed to polyphenols and alkaloid secondary metabolites viz., rutin, psoralen, limonene, pinene, coumarins, furanocoumarins and acridone (Elansary et al. 2020).

Phenolics constitute major antioxidant compounds that protect the cells from damaging effects of reactive oxygen species (ROS) generated normally in cells during metabolism. ROS have implications in several health disorders and the current knowledge on the relevance of antioxidant compounds in the fight against pathogens, commuter diseases, diabetes, cardiovascular, neurodegenerative diseases like Parkinson’s and Alzheimer’s disease (Lima et al. 2019) makes them high demand compounds in pharmaceutical and cosmetic industries. Natural antioxidants have attracted much attention because of the increasing awareness about their safety and efficacy. This makes industries heavily dependent on natural sources and plants in particular. The useful compounds are obtained by extraction from the plant that threatens its conservation. Further, secondary metabolites are generally low bulk compounds and therefore the quantity harvested is not sufficient to meet the ever-increasing demand. In vitro plant tissue cultures have proved to be a good alternate and perpetual source for the production of secondary compounds (Chandran et al. 2020). In recent years researchers have focused on the biotechnological production of useful compounds by employing elicitation strategy. In this, biotic and abiotic molecules are added in small concentrations into the growth medium which then attaches to special receptors located on plant cell membranes, enters into the cell and triggers signal transduction cascade which leads to transcriptional reprogramming of genes encoding enzymes of secondary metabolite biosynthesis pathways (Ramirez-Estrada et al. 2016). This strategy has been reported to enhance the yield of secondary metabolites in Gynura bicolor (Shimizu et al. 2010), and Knautia sarajevensis (Karalija et al. 2020).

Methyl jasmonate (MJ), a naturally occurring fragrant volatile ester form of jasmonic acid, is known to work as signal transducer to stimulate plant defense mechanisms by regulating secondary metabolism (Benevenuto et al. 2019). Given this role of MJ, it has been suggested that MJ could be used as a chemical elicitor (Rubio-Rodríguez et al. 2021). Abscisic acid (ABA) is an isoprenoid phytohormone. It regulates many aspects of plant growth and response to environmental stress. It is reported to induce the expression of genes involved in phenylpropanoid biosynthetic pathway leading to the accumulation of defense metabolites (Gai et al. 2020).

R. graveolens is scarcely available in nature and its cultivation is also little due to lengthy and insufficient propagation methods. Given its immense medicinal value several workers have developed its tissue cultures for conservation and metabolite production (Bohidar et al. 2008). There are many reports on elicitation of coumarins and furanocoumarins in R. graveolens (Ahmad et al. 2020). However, as per the literature no systematic studies have been conducted to improve polyphenolic compounds in R. graveolens except for the one which demonstrated the role of different light conditions on accumulation of phenolics in shoot cultures (Szopa et al. 2012).

Active compounds of R. graveolens L. are primarily concentrated in stem and leaves. Therefore, we established its shoot cultures and attempted to apply an elicitation strategy to improve polyphenol biosynthesis. For this purpose, we used MJ and ABA as elicitors. The aim of the study was to test the efficacy of MJ and ABA on the accumulation of polyphenolic compounds and antioxidant potential in shoot cultures of R. graveolens. We also examined the effect of elicitor exposure period as this factor is known to influence the efficiency of the elicitor.

Materials and methods

Plant material and in vitro shoot culture

Ruta graveolens L. plants maintained at the Department of Dravyaguna Herbal Garden, School of Ayurveda, D.Y.Patil Deemed To Be University, Navi Mumbai, India were used for the collection of explants. Nodal segments of R. graveolens were washed under running tap water for 5 min, surface sterilized with 0.1% mercuric chloride (w/v) for 5 min followed by thorough washing with distilled water and inoculated on culture initiation medium i.e. MS medium (Murashige and Skoog 1962) containing 3% (w/v) sucrose, 0.8% (w/v) agar, 1.0 mg L–l 6-benzyl aminopurine (BAP) and 0.25 mg L–l Indole acetic acid (IAA) (Bohidar et al. 2008). Cultures were incubated at 25 ± 2 ◦C, under a 16 h photoperiod of light intensity of 45 µMol m− 2s− 1 provided by cool white fluorescent tubes. The shoots obtained were multiplied by subculturing thrice at an interval of 21 days on the same fresh medium in culture bottles. The multiplication phase shoot cultures were used as the experimental material (Fig. 1).

Fig. 1
figure 1

In vitro shoot culture of R. graveolens

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Elicitation treatments

A shoot cluster weighing 0.5 g (containing ca. 8 shoots measuring 0.5 cm) was inoculated on culture initiation medium supplemented with: (a) methyl jasmonate (100 µM), (b) abscisic acid (20µM) and (c) no elicitor (control), six replicates each. For each treatment and control, shoots were harvested on 5th, 10th and 15th day of elicitation for growth and biochemical studies. Growth of shoots was determined in terms of fresh weight (FW) (g), number of shoots per cluster and shoot length (cm).

Biochemical studies

Extract preparation

For biochemical studies, extracts from shoots of control and elicited cultures were prepared as described by Mihai et al. (2011) with slight modification. A methanol: 0.1% HCl 80:20 (v/v) solution was used as a solvent. The sample to solvent ratio was 1:2. The sample paste was suspended in a conical flask and subjected to ultra-sonication at a frequency of 47 kHz using an ultrasonicator model No. 5.5 L 150 H (Dakshin Ultrasonics, India). The extract obtained was kept overnight at 40 °C in the refrigerator for 12 h. The insoluble material was removed by centrifugation at 4000 x g for 15 min (Superspin R-V/Fa, Plasto crafts, India) and the extracts were concentrated by evaporation using rotavapor (Rotary Vacuuma ‘Digital Bath’, Superfit Continental Pvt. Ltd. India) and used for biochemical studies as described below:

Total phenolic content

The total phenolic content (TPC) was determined using the Folin Ciocalteu (FC) method described by Supritha and Radha (2018). 0.5 ml of sample extract (1 g L− 1) was added to 2.5 ml 10-fold diluted FC reagent and 2 ml 7.5% sodium carbonate (Na2CO3). The tubes were kept in dark at room temperature for 30 min followed by measurement of absorbance at 760 nm against a blank UV visible spectrophotometer (UV-1700 Pharma Spec, Shimadzu). The total phenolic content was determined from the standard curve of gallic acid and expressed as mg gallic acid equivalent (GAE) g− 1 FW.

Total flavonoid content

The aluminium chloride colorimetric assay was used to measure the total flavonoid content (TFC) of the plant extracts (Marinova et al. 2005). 1 ml of the extract (1 g L− 1) or standard (quercetin) was added to a 10 ml volumetric flask containing 4 ml of distilled water. 0.3 ml of 5% sodium nitrite (NaNO2) was added to the flask and after five minutes, 0.3 ml of 10% aluminum chloride (AlCl3) was added. The volume was made up to the mark with distilled water. The solution was mixed and allowed to stand for 30 min at room temperature. The absorbance of reaction mixture was measured against the blank at 510 nm using a UV visible spectrophotometer. The total flavonoid content was quantified from the standard curve of quercetin and expressed as mg quercetin equivalents (QE) g− 1 FW.

Total flavonol assay

The aluminium chloride method described by Akkol et al. (2008) was followed for the estimation of total flavonol (TFL) content. 2 mL of shoot extract (1 g L− 1) was mixed with 2 mL (20 g L− 1) AlCl3 and 6 mL(50 gL− 1) sodium acetate (Akkol et al. 2008) followed by incubation at 20 °C for 2.5 h. The absorbance of the reaction mixture was recorded at 440 nm. Total flavonol content was measured from the standard curve of rutin and expressed as mg rutin equivalents (RE) g− 1 FW.

Phenylalanine ammonia-lyase (PAL) activity assay

Fresh shoots (1 g) were homogenized in 10 mL Tris-HCl (pH 8.2, 100 mM) buffer containing β-Mercaptoethanol (15 mM). The mixture was centrifuged at 10,000 x g, 4 °C for 15 min and the clear supernatant was used as enzyme extract. The reaction mixture containing enzyme extract (0.5 mL), 16 mM of L-phenylalanine, 50 mM of Tris–HCl buffer (pH 8.9), and 3.6 mM of NaCl was incubated at 30 °C for 60 min and then the reaction was stopped by adding 50 µL of 5 N HCl. The PAL activity was determined by measuring the initial (0 min) and final absorbance (60 min) of the reaction solution at 290 nm (Dong et al. 2010). The amounts of cinnamic acid produced within 1 h per mg of protein were used to express one unit of PAL activity. Total protein estimation was carried out according to Bradford assay (1976) using bovine serum albumin as the standard.

Analysis of antioxidant potential

Antioxidant activity was determined using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay (FRSA) and Ferric ion reducing antioxidant power assay (FRAP) assay.

For DPPH-FRSA assay, protocol described by Vijayaraghavan et al. (2013) was followed using butylated hydroxy toluene (BHT) as standard. The ability of extracts to scavenge DPPH radical was assessed and antioxidant activity was expressed as % DPPH inhibition calculated using the formula:

DPPH-FRSA (% inhibition) = Abs of control – Abs of sample/ Abs of control *100.

Where, Abs control is the absorbance of the control (standard) and Abs sample is the absorbance of the test sample (extract).

FRAP assay was performed using the method described by Benzie and Strains (1996). The antioxidant activity based on ferric ion reducing ability was calculated using a standard curve of ascorbic acid at 593 nm. The FRAP result was expressed as mg of ascorbic acid equivalent antioxidant capacity g− 1 FW (mg AEAC.g− 1 FW).

HPLC analysis

Quantification of phenolic compounds was performed for shoots from control and those treatments which showed maximum TPC (MJ-5 d and ABA-15 d post elicitation) using HPLC (Waters Model 2487 with UV detector). The stationary phase was C18 column (5µM). The standards of gallic acid, catechol, caffeic acid, ferulic acid, p-coumarin and vanillin were prepared in methanol (1000 ppm) and diluted to 50 ppm. The elution system was composed of 20% methanol, 1% acetic acid and 80% water with a linear gradient scheme and the detector was set at 280 nm (Ranade et al. 2016). For quantification of rutin, the standard was prepared in methanol (1000 ppm) and diluted to 100 ppm. The mobile phase consisted of methanol–water,1:1 (v/v) (pH 2.8 adjusted with phosphoric acid) with isocratic scheme and absorbance was monitored at 360 nm (Kuntić et al. 2007). Methanolic extracts were used for HPLC and the phenolic compounds and rutin were identified on the basis of their retention time on comparing with the standard chromatogram of a mixture of pure phenolic compounds and rutin, respectively. The concentration of compounds was determined from the peak area measurement and was expressed as mg g− 1FW.

Statistical analysis

Experiments were conducted in randomized design; results were expressed as mean values (± standard deviation, SD) with three replications. Each treatment was tested for two-way ANOVA assuming no sphericity with 95% confidence interval. Tukey’s multiple comparisons test was further applied to validate the difference between means of each treatment. All statistical testing and graphs plots were done using Graph Pad Prism 8.0.1.

Results and discussion

R. graveolens shoot cultures are known to synthesize polyphenolic compounds. The present study examined the effect of exogenous addition of MJ and ABA on accumulation of these metabolites in shoot cultures of R. graveolens. Analysis of variance (ANOVA) revealed that elicitor type, its contact time and the interaction between elicitor and contact time significantly influenced all the examined parameters.

Effect of elicitors on shoot growth

ABA is generally considered as a growth retardant (Rai et al. 2011) with few exceptions that suggest its role in promotion of shoot growth. MJ is also known to inhibit growth by suppression of cell proliferation and expansion or disruption of meristematic activity (Kamińska, 2021) and has shown negative effects on biomass accumulation in in vitro cultures of Gynura bicolor (Shimizu et al. 2010). Contrastingly, in the present study both the elicitors did not reduce any of the growth parameters but showed a slight increase (1.1-1.3-fold) upon long duration exposure. A maximum increase of 1.3-fold was seen in fresh weight and shoot number on ABA on 15th day post elicitation, compared to control (Table 1). Studies have revealed that growth-inhibiting effects of exogenous ABA can be counteracted by cytokinins present in the medium (Rai et al. 2011) and this might explain the slight increase seen in the present study. There are reports where MJ addition in the medium increased in vitro growth like shoot proliferation in Musa acuminata (Mahmood et al. 2012), shoot length in Ziziphora persica (Zare-Hassani et al. 2019, plant height in Artemisia annua (Alam and Albalawi 2020; Avalbaev et al. 2016) reported the role of MJ in increasing endogenous cytokinins level which affects cell division and growth. The differential effect of exogenous elicitors may arise from modification of synthesis, catabolism, activation, sequestration, transport, or sensitivity to endogenous phytohormones of the same or other type (Gaspar et al. 1996).

Table 1 Effect of elicitors on growth parameters of in vitro shoot cultures of R. graveolens
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Effect of elicitors on total phenolic content (TPC), total flavonoid content (TFC) and total flavonol content (TFL)

TPC, TFC, TFL are the most common markers of antioxidant potential. In the present study, both the elicitors increased TPC compared to control, and the highest value of 44.33 mg GAE g− 1 FW was obtained on MJ on 5th day post elicitation, representing a 4.4-fold increase over control followed by a 3.5-fold increase on ABA on 10th day post elicitation (Fig. 2). With the increase in exposure time, TPC declined on MJ but remained constant on ABA. Maximum accumulation of phenolics after short exposure of elicitor has been reported in K. sarajevensis (Karalija et al. 2020).

Fig. 2
figure 2

Effect of elicitors on total polyphenol concentration (TPC) and PAL activity in shoot cultures of R. graveolens. Values are means ± standard deviation of six replicates,*** and ** indicates the significant difference at p < 0.001 and p < 0.01, respectively, Tukey’s test

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On MJ, TFC was similar to control (0.26 mg QE g− 1 FW) on 5th day post elicitation but increased by 4-fold (0.87 mg QE g− 1 FW) on 10th day and remained constant thereafter (Fig. 3). This is in agreement with elevated TFC levels observed in in vitro cultures of Momordica charantia (Chung et al. 2016) elicited with MJ. TFC increased most by 6-fold (1.43 mg QE g− 1 FW) over control in shoots treated with ABA for 5 days and it then decreased by 104%with increase in exposure time. According to Piątczak et al. (2016), decline in metabolite content with increased elicitor contact might be the result of either metabolites degradation in situ or its leakage into the medium. Similar to our observations, exogenous application of ABA increased the phenolics and flavonoid contents in Tea (Gai et al. 2020). Recently, the transcriptomic and metabolomic studies revealed that exogenous ABA induced the expression of genes involved in flavonoid biosynthesis (Gai et al. 2020). Authors explained that on ABA, increase in biomass results in nutrient stress which stimulates the synthesis of antioxidant phenolics.

Fig. 3
figure 3

Effect of elicitors on total flavonoid concentration (TFC) and total flavonol concentration (TFL) in shoot cultures of R. graveolens. Values are means ± standard deviation of six replicates,*** and ** indicates the significant difference at p < 0.001 and p < 0.01, respectively, Tukey’s test

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Flavonols are a type of flavonoid compound. Highest TFL (1.22 mg RE g− 1 FW) was obtained in shoots elicited with ABA for 15 days, representing 2-fold increase compared to control (0.52 mg RE g− 1 FW). MJ also increased flavonol content by nearly 2-fold on 5th and 10th day post elicitation (Fig. 3). On ABA treatment, TFL increased with increase in exposure time while a reverse trend was seen for TFC. This indicates that it’s not flavonol but other flavonoids that contributed to the high TFC on the 5th day but its contribution increased with increase in exposure time. On MJ treatment, both TFC and TFL increased with exposure time. These results suggest that each compound may be differently influenced by a particular elicitor.

Both MJ and ABA are involved in plants’ response to stress by regulating the synthesis of defense molecules. They initiate de novo transcription of genes encoding enzymes of the biosynthetic pathways of phenolics and flavonoids (Gai et al. 2020). Jalalpour et al. (2014) showed that increased phenolic metabolism in MJ elicited cells was due to enhancement of endogenous jasmonate levels through induction of lipoxygenase (LOX) activity. It is clear from our results that both elicitors displayed different effects on phenolics and flavonoids accumulation which could be attributed to the difference in the mode of action of the two phytohormones. They may induce different signal transduction pathways ultimately resulting in accumulating metabolites in different concentrations. These concentrations also varied with elicitor contact period which could be because for each biosynthetic pathway the time required for its enzymes to show maximal activity is different (Piątczak et al. 2016).

Effect of elicitors on PAL activity

Polyphenols are produced through the phenylpropanoid pathway and this is the most analyzed pathway in elicitation studies. Biosynthesis of phenolics and flavonoids initiates through deamination of L-phenylalanine to trans-cinnamic acid by the enzyme PAL (Sharma et al. 2019). In the present study, shoots treated with MJ and ABA showed 10-fold (2.8 µM CA min− 1.g− 1 protein) and 7.6-fold (2.0 µM CA min− 1.g− 1 protein) increase in PAL activity, respectively compared to control on 5th day post elicitation (Fig. 2). On both elicitors, the activity declined with exposure time, the effect however was more significant on MJ. Increased PAL activity was also in correspondence with increased TPC and TFC in both MJ and ABA treated shoots, except for TFC on MJ on 5th day post elicitation. Increased accumulation of these metabolites is usually associated with increased activity of PAL (Rubio-Rodríguez et al. 2021). Our results agree with the role of this enzyme in antioxidant metabolite synthesis.

RP-HPLC quantification of phenolics and rutin

RP-HPLC was performed for some of the polyphenolics to see their individual contribution to TPC and TFC. It was carried out for only those treatments which showed maximum TPC (5 days MJ treated and 15 days ABA treated shoots). It was observed that MJ induced accumulation of all the analyzed phenolics viz. gallic acid, catechol, caffeic acid, p-coumaric acid, caffeic acid, vanillin and ferulic acid while ABA treated shoots showed the presence of only p-coumaric acid, vanillin and caffeic acid (Table 2, Supplementary information). Control shoots showed the presence of vanillin at the concentration of 24.1 mg.g− 1 and 30.0 mg.g− 1 on the 5th and 15th day of incubation, respectively. Its accumulation increased by 198%(72.0 mg.g− 1) and 32% (39.6 mg.g− 1) on MJ (5 days) and ABA (15 days), respectively. p-coumaric acid was also detected in control shoots on the 15th day (9.5 mg.g− 1) and that was increased by 2-fold and 8-fold on MJ and ABA, respectively. The absence of other phenolics in control shoots could be because their synthesis was not within the detectable limits. In the present study, presence of additional phenolics in elicited shoot cultures indicates the role of elicitors in the regulation of their biosynthetic pathways. MJ-mediated elicitation of phenolic compounds has also been reported in in vitro cell cultures of Vitis vinifera (Sák et al. 2014).

Table 2 RP-HPLC based quantification of phenolic compounds and rutin in R. graveolens L. shoot cultures treated with elicitors
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Rutin was quantified in shoots from control and those treatments which showed maximum TFL (MJ-15 d and ABA-15 d post elicitation). In control shoots, rutin was detected at a concentration of 34.5 mg.g− 1. Its accumulation increased by 3.5-fold and 2.5-fold in shoot treated for 15 days with ABA and MJ, respectively (Table 2). This increase was in correspondence with TFL indicating substantial contribution of rutin in TFL.

Effect of elicitors on antioxidant activity (DPPH & FRAP)

The protective effect of phenolics and flavonoids are attributed to their antioxidant capacity. It was determined using widely accepted assays that measure different antioxidative ability like free radical scavenging (DPPH assay) and transition metal ion reduction (FRAP assay). In response to both elicitors, shoots showed higher DPPH-FRSA compared to control indicating synthesis of DPPH scavenging antioxidant compounds (Fig. 4). The highest value of DPPH inhibition (82%) was recorded on ABA on 10th day post elicitation which was 2-fold higher than control (42.33%) and it declined to 78% with increased duration of exposure. On MJ, DPPH inhibition was 21.9% which was less than control (36.86%) on 5th day post elicitation but was higher than control on long exposure, representing a 13% increase over control (42.26%).

Fig. 4
figure 4

Effect of elicitors on antioxidant potential; DPPH activity (% inhibition) and FRAP value in shoot cultures of R. graveolens. Values are means ± standard deviation of six replicates,*** and ** indicates the significant difference at p < 0.001 and p < 0.01, respectively, Tukey’s test

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Antioxidant activity measured by FRAP assay increased and decreased with duration of exposure of ABA and MJ, respectively. The highest FRAP value of 0.4 mg AEAC g− 1 FW was obtained on MJ on 5th day post elicitation, representing an increase of 1.5-fold followed by ABA treatment for 15 days which showed a 1.7-fold increase over control (Fig. 4).

Wee et al. (2015) also reported a 1.3-fold increase in antioxidant activity (DPPH and FRAP) in MJ elicited-callus cultures of Sauropus androgynous. ABA showed high FRAP value (1.2 fold) than control (0.29 mg AEAC g− 1 FW) on the 10th day of exposure which supports the previously realized fact that ABA can stimulate the expression of antioxidant genes thus enhancing the antioxidant capacity in plants (Choudhary et al. 2012).

On comparing the results of two assays it was found that both DPPH inhibition and FRAP values increased with exposure time of ABA but on MJ the two parameters showed a contrasting trend. Also, the increase in antioxidant capacity measured by DPPH was more than FRAP for both elicitors. This difference could be because the two methods have different principles and mechanisms to measure antioxidant capacity. Comparison of antioxidant capacity and antioxidant compound contents revealed that results of DPPH-FRSA correspond with TPC and TFC in case of ABA and MJ elicitation, respectively. On both ABA and MJ, FRAP values corresponded with TPC but not with TFC indicating more contribution of phenolics than flavonoids in reducing ferric ions. It is known that those antioxidants that react quickly with transition metal ion will react slower or may not react with DPPH due to steric hindrances while some reports have shown that a combination of polyphenolic compounds produced a synergistic effect on DPPH and FRAP (Khorsidi and Nijavan 2006).

Conclusion

Elicitors increased polyphenolics accumulation and antioxidant potential in shoot cultures of R. graveolens L. Exposure time of elicitor also played a significant role in metabolite synthesis. Maximum accumulation of TPC and TFC was obtained after short time exposure of MJ and ABA, respectively. Shoots treated with MJ for 5 days showed the presence of all the phenolics estimated by HPLC. Both the elicitors improved metabolite production without compromising on biomass growth. These findings suggest that shoot cultures of R. graveolens could be used as a potential source of polyphenolic compounds with antioxidant activity which are in great demand in pharmaceutical, cosmetic and food industries.