A Study On The Effect of Different Elicitors On Capsaicin Accumulation in Cell Suspension Cultures of Capsicum Assamicum (Bhut Jolokia)

Elicitation of cell suspension cultures of Capsicum assamicum (Bhut Jolokia) for enhancement of capsaicin content was tried using different elicitors such as cellulase, vanillin, methyl jasmonate, salicylic acid and sinapic acid in different concentrations for 24, 48 and 72 hours. Cell suspension culture was established in B5 media supplemented with 3.5 mM 2,4-D (2,4-diphenoxyacetic acid) and 1.1 mM Kin and elicitors were introduced at the end of exponential phase. All the elicitors, except methyl jasmonate, led to signicant increase in production of capsaicin. Sinapic acid, when added in 22 µM concentration and incubated for 24 hours, led to highest capsaicin accumulation of 0.5% (5068 µg/g) which was highest among all the treatments. The results were found to be statistically signicant with p < 0.0001. the studies conducted by Pandhair and Goyal it was found that on addition of sinapic acid at 44 µM concentration to the cultures of Capsicum annum L., there was 8.9 fold increase in capsaicin accumulation. The results were found to be statistically signicant with p < 0.0001.


Introduction
Capsicum assamicum (Bhut Jolokia), a chilli species endemic to North East India, is one of the hottest chillies in the World with a Scoville Heat Unit (SHU) of more than 1 million (Guinness Book of World's Record, 2013). Capsaicin, the compound responsible for its hotness, has numerous applications in food and pharmaceutical industries. It is generally extracted from the chilli fruit. However, most Indian chilli species produce less than 1% capsaicin, C. assamicum, which contain 3-5% capsaicin, is a suitable candidate for commercial production, but the chilli fruit is available only from May to October (Borgohain and Devi, 2007). Cell suspension culture can be a suitable alternative for large scale production of capsaicin, round the year and independent of climatic and geographic conditions. No report regarding the use of suspension culture for capsaicin production from C. assamicum could be traced. With this aim, callus had been induced and cell suspension culture has been established (Swetnisha et al., 2018). However, the amount of capsaicin accumulated was found to be very low.
Elicitation is the process of enhancement of a particular compound of interest in cell cultures by addition of a foreign material (Veersham, 2004). Previously conducted studies have shown that elicitors increase secondary metabolite accumulation in plant cell cultures (Brooks et al., 1986). The present study investigates the use of different elicitors, viz. cellulase, salicylic acid, sinapic acid, vanillin and methyl jasmonate, on accumulation of capsaicin in suspension cultures of C. assamicum.
Cellulase is an enzyme produced by a wide range of microbes including bacteria, fungi and protozoa that plays an important role in the hydrolysis of lignocellulosic materials (Jayasekara and Ratnanayake, 2019). Being of microbial origin, it mimics microbial infection and may trigger defence response resulting in capsaicin production. Sinapic acid is a phytochemical which belongs to a class of phenolic acids known as hydroxycinnamic acid (Chen, 2015). It is a chemical analogue of caffeic acid which is a precursor in capsaicin biosynthetic pathway. Hence, it is worth investigating the effect of sinapic acid on capsaicin production. Vanillin is also a precursor in phenylpropanoid pathway of capsaicin biosynthesis.
Hence, its addition in cell cultures might enhance capsaicin production. Salicylic acid (SA) is a derivative of chorismate which is a signal molecule in plants and act as an inducer of systemic acquired response (SAR) against pathogenic attack (Ryals et al., 1996). In the event of pathogen attack, level of salicylic acid has been found to increase (Verberne et al., 2000;Mustafa et al., 2009). Since capsaicin is synthesized as a defence mechanism, introduction of salicylic acid might induce SAR which would lead to the production of capsaicin. Methyl jasmonate (MeJA) is a commonly used elicitor. It is known to have both inhibitory and stimulatory effect, physiologically as well as morphologically. It also induces defence metabolism in cultured cells (Gundlach et al, 1992;Sembdner and Parthier, 1993;Belchert, 1995). Enhancement in alkaloid production after treatment with MeJA has also been shown in a number of plant cultures (Aerts et al., 1992;Gundalch et al., 1992;Zabetakis, 1999). In the present study, the effect of all

Elicitation of cell cultures
Cellulase, vanillin, sinapic acid, salicylic acid and methyl jasmonate were used for culture elicitation. All the elicitors were obtained from Sigma-Aldrich. The elicitor solutions were lter sterilized using sterile syringe lter of 0.2 µm pore size under Laminar Air Flow. At the end of the exponential phase, elicitors were added to the cultures in different concentration and incubated for 24, 48 and 72 hours. in the same culture condition as before. Unelicitated cultures were used as control. All the experiments were performed in triplicates.

Capsaicin extraction and quanti cation
For extraction of capsaicin, callus was ltered from the suspension culture using Whatman No. 1 lter paper and washed with distilled water. The callus was collected in a pre-weighed petridish and dried in circulatory hot air oven at 50 O C. Dry weight of the callus was recorded and was crushed into ne powder. Capsaicin was extracted in 80% methanol using cold maceration. Since capsaicin is stored extracellularly, it often gets leached into the media as well. Hence, capsaicin was extracted from media using the method used by Johnson et al., 1992. Media was washed with ethyl acetate in a separating funnel in 2:1 ratio. The upper layer of ethyl acetate was collected. The process was repeated thrice with the remaining media. The extracts were pooled and evaporated to obtain dry extract residue and stored at -20 O C until further processing. Capsaicin quanti cation was done through HPLC based on the protocol standardized by Gonzales-Zamora et al., 2015. Mobile phase for HPLC constituted of an isocratic mixture of water: acetonitrile (50: 50) and 20 µl injection volume was eluted. Detection was done using UV detector at 280 nm. Capsaicinoid related peak was obtained within 20 minutes. Capsaicin content of the extracts was quanti ed by plotting the peak area of chromatogram on the standard curve prepared on the basis of HPLC chromatogram of standard capsaicin (Fig. 1).

Statistical Anlysis
Data were analyzed using Two Way ANOVA and sample means were compared via Tukey's HSD test at p < 0.05 using GraphPad Prism 8.3.0 software package.

Results And Discussion
All the ve elicitors used were shown to have different effect on capsaicin accumulation when used in different concentrations for different time period. The effect of different elicitors on capsaicin accumulation is described below: -Cellulase: Application of cellulase in cell suspension cultures of C. assamicum resulted in 14 times increment in capsaicin accumulation compared to untreated callus. Both cellulase concentration and incubation time affected capsaicin accumulation (Table 1). When cellulase was applied for 24 hours in the concentration 40 mM, highest capsaicin accumulation of 0.058% (5.84 µg/g) was observed followed by cellulase application in the same concentration for 48 hours and 72 hours respectively. It was found that the  Elicitation through salicylic acid was found to have positive effect on the capsaicin production in cell suspension culture of C. assamicum. Capsaicin production was found to be affected by both the concentration as well as the incubation period (Table 3). Highest capsaicin content of 0.016% (1.615µg/g) was found in cultures to which salicylic acid was added in 217µM concentration and incubated for 48 hours. Another study (Sudha and Ravishankar, 2003) showed that the highest capsaicin was produced (50 µg/g f.w of callus) in the cultures of Capsicum frutesenes treated with salicylic acid. In the present study, after treatment with salicylic acid, highest capsaicin content of 1.6 µg/g d.w of callus was observed. Of all the elicitors used, elicitation using sinapic acid showed the best result. Sinapic acid, in all the concentrations and for all the incubation period, enhanced the total capsaicin accumulation in the cultures of C. assamicum (Table 4). Highest capsaicin accumulation of 0.5% (5068.23 µg/g) was found in cultures to which sinapic acid was added in 22 µM concentration and incubated for 24 hours. In the studies conducted by Pandhair and Goyal (2009), it was found that on addition of sinapic acid at 44 µM concentration to the cultures of Capsicum annum L., there was 8.9 fold increase in capsaicin accumulation. The results were found to be statistically signi cant with p < 0.0001. Elicitation using MeJA did not have any signi cant effect on capsaicin production ( Table 5). Highest capsaicin production of 0.007% (0.716µg/g) was observed when it was added in 0.5 µM concentration for 48 hours as compared to control where 0.423µg/g capsaicin was produced. The results were not found to be statistically signi cant with p > 0.05. As the maximum capsaicin production was 0.7 µg/g (in cultures treated with 0.1 µM MeJA) as compared to 0.4µg/g production in untreated callus, it can be said that MeJA was not very effective in enhancing capsaicin accumulation. A study conducted by Sudha and Ravishankar (2003) showed that the addition of 0.05 µM MeJA to the cultures of Capsicum frutesenes when incubated for 12 days, resulted in 40 µg/g f.w of callus. When MeJA was applied in the same concentration, it was found that capsaicin production was 0.7 µg/g d.w of callus. It is di cult to compare the two results as analysis was done in fresh weight in former while it was done in dry weight in latter.

Conclusion
Capsaicin is an important industrial and pharmaceutical product, which is generally extracted from different members of the Capsicum genus. Though arti cial production through chemical and enzymatic synthesis is in use, there are certain limitations. Chemical synthesis employs a number of reagents and catalysts and releases certain by-products, that are known to be toxic, which is a major drawback. Enzymatic synthesis is free from toxic reagents and by-products; however, the total yield of capsaicin through this method is very low. The only viable method for commercial production is extraction from plants. As the criteria for commercial production clearly dictates that the source plant must contain more than 1% capsaicin and none of the Indian chillies, except Bhut Jolokia, contain more than 1% capsaicin. But the major issue is the limited cultivation of this chilli in few states of North East India and its seasonal nature of production. Thus, plant tissue culture technique can ensure round the year production of capsaicin.
Based on above results, it can be concluded that sinapic acid and vanillin showed promising effect on capsaicin accumulation. Both elicitors have enhanced yield of capsaicin in cell cultures by factor of hundreds (Tables 2 & 4). Vanillin, being an intermediate in capsaicin biosynthetic pathway, acts as a potent elicitor. The results showed that sinapic acid has been, by far, the best elicitor which led to highest capsaicin yield of 0.5% compared to all other elicitors used in this study. In earlier studies also, both these elicitors have shown promising response and led to signi cant increase in capsaicin accumulation (Pandhair and Goyal, 2009).
Although signi cant enhancement in capsaicin accumulation has been observed, there are a number of other methods that can be studied to further enhance its production. Effect of various intermediates and precursors of capsaicin biosynthetic pathway on its production through suspension culture needs to be investigated. Besides, biotransformation and use of transgenics, for enhancing capsaicin production can also be explored.