Effects of Sequential and Simultaneous Double Elicitation on Aloe-Emodin Production in Cassia Tora Root Cultures

Cassia tora root cultures were established to study anthraquinone production and as an alternative source of anthraquinone. In this study, several plant defence-based techniques were used to enhance aloe-emodin production in Cassia tora root cultures. Elicitation and the initial optimisation of exposure time and culture age suggested that 100 mg/L yeast extract was the most ecient elicitor, with the highest eciency during early to mid-stationary growth phase. The elicited root produced approximately 2.7-2.9 folds-improvement in aloe-emodin content when compared with the control. The eciency of the elicitor tended to increase along with the exposure time. Extending the elicitation period to the end of culture cycle (9-10 days) increased the production up to 6.21 times (5.740 ± 0.584 mg/gDW). Simultaneous double elicitation and sequential double elicitation also showed their effectiveness on aloe-emodin production. Aloe-emodin production was improved up to 9.09 times higher than the control by a simultaneous add-on of 50 mg/L chitosan with 100 mg/L yeast extract. Meanwhile, sequential elicitation of 50 mg/L chitosan for 24 hours prior to elicitation by yeast extract improved the production up to 11.27 times higher than the control. Both treatment styles exhibited a particular advantage toward the extracellular accumulation, which might facilitate the use of two-phase culture or in conjunction with other enhancing techniques.


Introduction
Plants have profound impact on human lives. One important role is supplying pharmaceutically-value compounds. For examples, khellin from Ammi visnaga is a lead compound of the asthma prophylactic drug, sodium cromoglycate. Paclitaxel from Taxus sp. as well as vinblastine and vincristine from Catharanthus roseus have been used as chemotherapy for various types of cancer (Rungsung W et al., 2015). Aloe-emodin is an anthraquinones reportedly produced by Cassia tora (Kim YM et al., 2004) and has been studied as a candidate for an anticancer drug for decades (Özenver N et al., 2018). Other than the laxative effect which is the hallmark of anthraquinones, the compound has been reported to exhibit cytotoxic activity toward various cancer cell lines without causing undesirable effects on normal cells (Liu K et al., 2012;Pecere T et al., 2000). Apart from the pharmaceutical investigation, the development of an aloe-emodin supply source is undeniably a vital part of drug research and development, since the commercial price of aloe-emodin is still relatively high. Moreover, the chemical synthesis might not be cost-effective due to the involvement of extreme reaction conditions, heavy metal catalysts, and a series of reactions (Franck H-G, 1988).
Plant tissue cultures have become an alternative source of natural products. They have been widely used, not only as a model to study secondary metabolite production, but also as an industrial scale production source of some naturally occurring drugs (Bringi V et al., 2012). In spite of this, the major drawback of natural sources is low production level, since secondary metabolites are mainly produced as defence response against stress conditions (Wilson SA and Roberts SC, 2012). Elicitation is a current technique to increase secondary metabolite production. It is based on triggering plant defence systems by using biotic or abiotic stress. The e ciency of elicitation depends on various factors, such as plant species, elicitor concentration, exposure time and growth stage. Optimisation of these factors reportedly improves the elicitation outcome (Naik PM and Al-Khayri JM, 2016).
Chitosan and yeast extract are well-known fungal elicitors. The studies in Cassia sp. as well as other species have consistently suggested their effectiveness in anthraquinone production ( Vascosuelo A., et al., 2003). Moreover, a number of studies have suggested the advantages of using more than one enhancing technique in combination. Elicitation has been reported to synergistically work with many techniques, for example, precursor feedings (Sivanandhan G et al., 2014) and two-phase cultures (Byun SY et al., 1992). Simultaneous double elicitation is another technique to improve elicitation e ciency. It is based on the synergistic effect of two or more elicitors. The combination of methyl jasmonate and chitosan was reported to exhibit a dual elicitation effect (Al-Gendy AA and Lockwood GB, Primed state is an underlying defence protection suggested to be established in plants after interaction with pathogens. Plants with primed states are more sensitised to stress than usual and are able to produce more intense defence responses ( Our previous study showed that C. tora root cultures were able to be used as a potential alternative source of aloe-emodin (Sakunphueak A et al., 2013). In order to improve the yield of aloe-emodin in C. tora root cultures, effects of chitosan and yeast extract elicitation were investigated in this study. The factors that affect the outcome of elicitation were optimised and 2 treatment patterns including simultaneous double elicitation and sequential double elicitation of both elicitors were studied.

Establishment of C. tora root cultures
The root cultures were established from C. tora plantlets. The root explants were excised and cultured in Gamborg's B5 media supplemented with 0.1 mg/L 1-naphthaleneacetic acid and 1.0 mg/L kinetin. The root cultures were generated from the root explants, separated from the original root and subcultured every 21 days until homogenous. The roots were cultured under sterile and dark conditions at 25 ± 2 °C with continuous shaking at 60 rpm. The homogenous root cultures were subsequently supplied as plant material for the studies (Sakunphueak A et al., 2013).

Preparation of elicitors
Chitosan: the stock solution of chitosan was prepared from low molecular weight chitosan  (Sigma-Aldrich) with 75-85% degree of deacetylation. Chitosan powder was dissolved in a small amount of 2% acetic acid with sonication. The solution was then adjusted to the nal volume with distilled water, and sterilized by autoclaving (121 °C, 15 min).
Yeast extract: the stock solution of yeast extract (Himedia) was prepared by dissolving yeast extract powder in distilled water. The solution was sterilized by autoclaving (121 °C, 15 min).

Optimisation of elicitor type and concentration
The optimisation of elicitor type and concentration was performed in 12-day old C. tora root cultures. Chitosan was added to the culture media to make a nal concentration of 10, 50, 100 and 250 mg/L, while yeast extract was added to make a nal concentration of 10, 50, 100 and 1000 mg/L. Sterilizeddistilled water at the same volume as the elicitor solution was added to make the control. Each elicitor was left to contact with the root cultures for 24 hours. The elicited root cultures and the media were then harvested and collected for the extraction processes.

Optimisation of exposure time
The 12-day old root cultures were elicited with the optimised elicitor from 2.3.1, then left to contact with the elicitor for 12, 24, 36 and 48 hours. The control was treated with sterilized-distilled water at the same volume as the elicitor solution. Subsequently, the elicited root cultures and the media were harvested and collected for the extraction processes.

Optimisation of culture age
The optimised elicitor from 2.3.1 was used to elicit the 9, 12, 16 and 18 days-old C. tora root cultures, under the optimised elicitation period suggested by 2.3.2. The root cultures at the same age were treated with sterilized-distilled water at the same volume as the elicitor solution to make the control group. Then, the elicited root cultures and the media were harvested and collected for the extraction processes.

Simultaneous double elicitation and sequential double elicitation
Simultaneous double elicitation was performed in the 12 days-old C. tora root cultures, using a combination of various concentrations of chitosan (10, 50 and 100 mg/L) along with 100 mg/L of yeast extract. Sequential double elicitation was performed by pre-treating the 12-day old root cultures with chitosan for 24 hours prior to the 100 mg/L yeast extract elicitation. Chitosan was added into the culture media to make a nal concentration of 10, 50 and 100 mg/L. After 24 hours, yeast extract at 100 mg/L was added into the pre-treated root cultures. The code of each treatment group is described in Table 1.  The treated root cultures were maintained until the end of culture cycle, then harvested. The root cultures and the media were collected for the subsequent extraction processes.

Intracellular aloe-emodin
The root cultures were dried at 50 °C for 24-48 hours and ground into powder. The root powder (100 mg) was extracted with 5% HCl in methanol (30 mL) at 70-75 °C using re ux apparatus. The extract was then concentrated by vacuum evaporator at 40 °C and reconstituted with methanol (10 mL) before being subjected to HPLC quantitative analysis of aloe-emodin (Sakunphueak A et al., 2013).

Extracellular aloe-emodin
The culture media were acidi ed to pH 2 using 1N hydrochloric acid, to switch the protonated aloeemodin to the deprotonated form. The acidi ed media subsequently underwent the liquid-liquid extraction with ethyl acetate. Then, the organic solvent part was collected and concentrated by vacuum evaporation at 40 °C. The extract was reconstituted in methanol and subjected to the HPLC analysis (Sakunphueak A et al., 2013).

Quantitative analysis of aloe-emodin
The aloe-emodin content was analysed using a HPLC system (Shimudzu, Japan), with photodiode array detector, and an autosampler. The separation was conducted in a reversed-phase TSKgel ® ODS-80Tm C-18 column (5 um, 150 mm x 4.6 mm i.d.). The gradient mobile phase consisted of 70% methanol and 30% aqueous acetic acid (2% v/v). The injection volume was 10 µL. The separation was performed with a ow rate of 1 mL/min and the quantitative wavelength was set at 266 nm. The relative concentration of aloe-emodin was calculated from the calibration curve of aloe-emodin standard and expressed in terms of mg per gram of the dry root powder weight (mg/gDW) (Sakunphueak A et al., 2013).

Statistical analysis
The data analysis was performed through the application PSPP (version 1.2.0), using one-way ANOVA and the post-hoc Tukey HSD test. The con dence interval was set to be 95 or 99% (p < 0.05 or 0.01).  (Iyer I et al., 2011). In this study, the un-elicited root cultures began the production of aloe-emodin on day 12 of the cycle and reached the peak level on day 20. The varied concentrations of yeast extract and chitosan were used to enhance the production. The elicitation period was initially set at 24 hours to avoid cytotoxicity caused by long-time contact with the elicitor. The elicitation effect was found to be highly in uenced by the type and concentration of elicitor. At 10 mg/L, both elicitors failed to enhance the production. However, their effects gradually improved as the concentration increased. At 50 mg/L of chitosan, the production level was elevated, but still not better than the control. In comparison, 50 mg/L of yeast extract signi cantly increased the production by 1.68 times when compared with the control (0.391 ± 0.024 mg/gDW). The peak performance of chitosan was observed at the concentration of 100 mg/L. It increased aloe-emodin production from the control by 1.18 times (0.276 ± 0.007 mg/gDW). While, the peak effect of yeast extract was also observed at 100 mg/L but it yielded a higher level of aloe-emodin than chitosan did (Fig. 1). Yeast extract at 100 mg/L was the most effective elicitor, increasing aloe-emodin production up to 2.71 times (0.633 ± 0.048 mg/gDW) when compared with the control. Nevertheless, the concentration-dependent manner only applies to the particular range of concentrations. At relatively high concentrations, the effects of chitosan and yeast extract were dropped from their peak level. Furthermore, these elicitors differently affected the accumulation in both intracellular and extracellular compartments. Yeast extract promoted a comparable accumulation in both compartments, whereas chitosan had a distinct advantage toward the intracellular accumulation (Fig. 1).

Results And
The results suggested that C. tora root cultures were more susceptible to yeast extract than to chitosan. Interestingly, similar ndings were also observed in C. angustifolia (Chetri SK  According to the results, we assumed that the defence mechanism of C. tora root cultures might not be sophisticated enough to e ciently respond to chitosan. It might have chitinases with less a nity on chitosan or consist of less compatible defence components. Meanwhile, yeast extract is a mixture of fungal cell components. It could contain many components that can trigger the defence system. Thus, yeast extract showed to be more e cient than chitosan at the same concentration. Furthermore, chitosan was suggested to negatively affect the growth and cause overstressed conditions in root tissue (Lopez-Moya F et al., 2017; Overvoorde P et al., 2010). This might be another in uence that leads to the lower elicitation e ciency of chitosan in C. tora root cultures.
Both chitosan and yeast extract elicitations exhibited a concentration-dependent manner until reaching the peak level and then started to decline. This phenomenon might be explained in term of defence capacity limitation. High elicitor concentration may exceed the available number of PRRs as well as the capacity of other defence components. Moreover, overstressed conditions caused by high concentrations of pathogen elicitors might be toxic to the plant cell and decrease the overall production (Overvoorde P et al., 2010).

Effects of exposure time and culture age
In order to further optimise the elicitation conditions, exposure time and culture age were subsequently optimised. Our result was consistent with previous reports in Rudgea jasminoides (Oliveira MDC et al., 2007) and R. cordifolia (Ghatge S et al., 2014), suggesting that longer exposure time produced better elicitation e ciency. The highest production was observed when the root cultures were exposed to the elicitor for 48 hours. The aloe-emodin production was signi cantly increased from the control up to 2.93 times (0.713 ± 0.010 mg/gDW).
The culture age was another affecting factor to optimise. Without elicitation, the aloe-emodin production was drastically increased from the middle of the stationary phase to the decline phase of the growth cycle. Aloe-emodin accumulation started on day 12, gradually increased until reaching the peak level on day 20, and then plummeted after day 24. According to the production pattern, the optimisation of culture age was speci cally performed during the aforementioned period -day 9, 12, 16 and 18 represented lag phase, exponential phase, late linear phase and early stationary phase of growth. Interestingly, the 100 mg/L yeast extract elicitation showed to be effective at every studied culture age. Treatment on day 12 promoted the highest elicitation bene t, with the 2.16 time-improvement in aloe-emodin production (0.1802 ± 0.012 mg/gDW) but elicitation on this day affected the root cultures health. Meanwhile, the treatment on day 16 did not show signi cant change when compared with the control. Interestingly, elicitation on day 18 showed negative impact on aloe-emodin production, actually lowering the aloeemodin content to 55% of the control. (Fig. 3). The result indicated that the elicitation was the most e cient during the exponential growth phase. However, it was too early to harvest the root cultures immediately after the 48 hours contact period (day 14) because the root cultures were not fully grown. Thus, we decided to perform elicitation on day 12 and harvest the elicited root cultures at the end of the culture cycle (day 21) in the following experiments.

Simultaneous double elicitation on aloe-emodin production
In the present study, 10, 50 and 100 mg/L chitosan in combination with 100 mg/L yeast extract were used to elicit the root cultures on day 12. The aloe-emodin levels were observed at the end of the culture cycle (day 21). Without chitosan, 100 mg/L yeast extract elicitation (Y100) increased the end cycle aloe-emodin production 6.21 times (5.740 ± 0.584 mg/gDW). It was found that 10 and 50 mg/L chitosan in a combination with 100 mg/L (DC10Y100 and DC50Y100) increased the production 8.82 times (8.139 ± 0.241 mg/DW) and 9.09 times (8.389 ± 0.400 mg/gDW), respectively. In contrast, a combination of 100 mg/L chitosan and 100 mg/L yeast extract (DC100Y100) overstressed the root cultures and caused the aloe-emodin content dropped when compared with the other treatment groups (Fig. 4). With the presence of a relatively low concentration of chitosan, 100 mg/L yeast extract elicitation enhanced aloeemodin production better than as a single elicitor. Meanwhile, the presence of the relatively high concentration of chitosan might have competitively consumed the defence resources and negatively affected the viability of the root cultures. Therefore 100 mg/L chitosan and 100 mg/L yeast extract failed to make a synergistic elicitor duo. This part of study suggested that simultaneous double elicitation of 10 mg/L chitosan and 100 mg/L yeast extract were the most effective conditions for enhancing aloeemodin production in C. tora root cultures.  Katz VA et al., 1998). In this part of the study, we took advantage of the primed state to further improve the e ciency of 100 mg/L yeast extract elicitation. Chitosan showed the ability to trigger defence response in the studied root cultures. Thus, it was chosen as the priming inducer. Various concentrations of chitosan were added into the culture media on day 12 in order to prime the root cultures. After 24 hours, 100 mg/L yeast extract was added to elicit the root cultures. Aloe-emodin production was measured at the end of culture cycles. It was found that suboptimal elicitor concentrations of chitosan created an entirely different priming effect. Root cultures primed with 50 mg/L chitosan (PC50Y100) showed to be more sensitised to 100 mg/L yeast extract elicitation. However, root cultures primed with 10 mg/L chitosan (PC10Y100) were not able to elevate the aloe-emodin level when compared with 100 mg/L yeast extract elicitation alone. PC50Y100 was able to elevate the aloe-emodin production (10.401 ± 0.531 mg/gDW) up to 11.27 times higher than the control which was 1.81 times higher than using yeast extract alone. Interestingly, chitosan at 10 mg/L and 100 mg/L failed to activate the priming effect. Both root cultures primed with 10 mg/L and 100 mg/L chitosan (PC10Y100 and PC100Y100, respectively) did not improve aloe-emodin production when compared to single yeast extract elicitation (Fig. 5).
The result was consistent with many reports, suggesting that defence responses under the in uence of primed states are more intense than usual. The priming effect is highly in uenced by a concentration of priming inducer. As recently reported by the study in Plumbago indica root cultures, pretreatment with the effective elicitor concentration of chitosan (150 mg/L) failed to improve the elicitation effect of 100 mg/L yeast extract on plumbagin production (Jaisi A and Panichayupakaranant P, 2020). This strongly agrees with our study and emphasizes that an effective elicitor concentration of chitosan does not work as a priming inducer concentration for 100 mg/L yeast extract elicitation. Meanwhile, pretreatment with low concentrations of chitosan was reported to enhance the disease resistance against fungal infection in Pinus patula seedlings and was involved with the increased expression level of gene encoding phenylalanine ammonia lyase (PAL) (Fitza KNE et al., 2013). The improved disease resistance was suggested to be associated with the improved hydrogen peroxide level in oxidative burst (Paulert R et al., 2010). Moreover, low concentrations of systemic acquired-resistance (SAR) inducers such as benzothiadiazole, 2,6-dicholoroisonicotinic acid and salicylic acid were reported to work as priming inducers in Petroselinum crispum cell cultures (Katz VA et al., 1998;Kauss H et al., 1992). These studies support our nding that low concentration or suboptimal concentration of elicitor is required for priming and suggests that different plant species require different types and concentrations of priming inducers.
To date, the majority of priming study have been focusing on pathogen resistance outcome. Consistent Whereas in terms of secondary metabolites, the study is completely different and needs more intensive investigation. Recently, reports on secondary metabolite production under the in uence of primed state have been from parsley cell culture-based studies, using SAR-inducers as the priming inducers (Katz VA et al., 1998;Kauss H et al., 1992) and a recent study in P. indica cell cultures which used methyl-βcyclodextrin as the priming inducer (Jaisi A and Panichayupakaranant P, 2020). Our work is probably the rst to report the success of using chitosan as a priming inducer to improve secondary metabolite production. It suggests a new aspect of chitosan priming, since previous reports largely focused only on the effect on disease resistance.
The precise mechanism of priming is still vague. Nevertheless, current suggestions give priority to the preexisting accumulation of defence molecules (signalling compounds, inactive enzymes and hormones), primary metabolism remodelling and epigenetic modi cations. We assumed that these intracellular changes might be the factors that discriminate the priming effect of different concentrations of chitosan in this study. (Tugizimana F et al, 2018;Espinas N et al., 2016;Zhu QH et al., 2016;Rojas CM et et al., 2014;Conrath U, 2011;Jaskiewicz M et al., 2011). Chitosan at 10 mg/L might be insu cient to induce signi cant aloe-emodin production itself or to sensitise the root cultures. Thus, the production in the PC10Y100 was purely contributed by the effect of yeast extract. Likewise, 50 mg/L was insu cient for the signi cant elicitation in a 24 hours period, but it might be intense enough to trigger PTI and induce the root cultures to the primed state. The case of PC100Y100, which 100 mg/L chitosan worked poorly as priming inducer, could be explained in term of primary metabolism remodelling. It has been suggested that plants lose resources and energy during PTI induction due to metabolic changes, affecting functions required to maintain normal growth and living (Schwachtje J et al., 2018;Rojas CM et al., 2014). Furthermore, the balance of jasmonic acid and salicylic acid signalling pathways were also reported to be interfered, following PTI induction (Schwachtje J et al., 2018). In order to compensate the imbalance and supply the alternative resources for normal living as well as further defence responses, plants need to process primary metabolism remodelling (Schwachtje J et al., 2018;Rojas CM et al., 2014). Single elicitation of 100 mg/L chitosan signi cantly increased the aloe-emodin production. This implies that 100 mg/L chitosan was able to trigger PTI and lead to the depletion of cellular resources. Thus, the root cultures primed with 100 mg/L chitosan probably need some appropriate interval to process primary metabolism remodelling for the subsequent responses to 100 mg/L yeast extract. According to the result, it might be assumed that 24 hours was not enough time for the root cultures to fully process primary metabolism remodelling. Thus, priming with 100 mg/L chitosan for 24 hours prior to 100 mg/L yeast extract elicitation (PC100Y100) did not promote further improvement over the single treatment of 100 mg/L chitosan (C100). Furthermore, PAMPs have been known to promote negative effects on plant health. Chitosan, especially at high concentrations, was reported to negatively affect root growth and development (Overvoorde P et al., 2010), as well as interfere with expression patterns of phytohormones and cause overstress in root tissues (Lopez-Moya et al., 2017). Thus, exposing the relatively high concentration of chitosan following by yeast extract might cause root culture death, and possibly be involved with lower aloe-emodin production.

Conclusion
In conclusion, sequential double elicitation and simultaneous double elicitation were successful in elevating aloe-emodin production in C. tora root cultures. Yeast extract was the most appropriate elicitor, whereas chitosan was found to be a priming inducer when used in combination with yeast extract. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Effect of chitosan and yeast extract concentration on aloe-emodin production in C. tora root cultures.

Competing interests
Data are displayed in term of mean ± SD (n = 6). The different letters on the top of the bars represent signi cant differences (p < 0.05).

Figure 2
Effect of exposure time on aloe-emodin production in response to 100 mg/L yeast extract elicitation. Data are the mean ± SD (n = 6). The different lowercase letters on the top of the bars represent signi cant differences (p < 0.01).

Figure 3
Effect of exposure time on aloe-emodin production in response to 100 mg/L yeast extract elicitation.
Data are the mean ± SD (n = 6). The different lowercase letters on the top of the bars represent signi cant differences (p < 0.01).

Figure 4
Aloe-emodin production in response to simultaneous double elicitation using chitosan and yeast extract. Data are the mean ± SD (n = 6). The different lowercase letters on the top of the bars represent signi cant differences (p < 0.05) Figure 5 Aloe-emodin production in response to sequential double elicitation using chitosan and yeast extract.
Data are the mean ± SD (n = 4-6). The different lowercase letters on the top of the bars represent signi cant differences (p < 0.05).