Flavonoid and Leaf Gas Exchange Responses of Centella asiatica to Acute Gamma Irradiation and Carbon Dioxide Enrichment under Controlled Environment Conditions

The study was couducted to investigate the effects of gamma irradiation and CO2 on flavonoid content and leaf gas exchange in C.asiatica. For flavonoid determination, the design was a split split plot based on Randomized Complete Block Design (RCBD). For other parameters, the designs were split plots. Statistical tests revealed significant differences in flavonoid contents of Centella asiatica leaves between different growth stages and various CO2 treatments. CO2 400, G20 (400 = ambient CO2; G20 = Plants exposed to 20 Gy) showed 82.90% higher total flavonoid content (TFC) in the 5th week than CO2 400 as control at its best harvest time (4th week). Increasing the concentration of CO2 from 400 to 800 μmol/mol had significant effects on TFC and harvesting time. In fact, 800 μmol/mol resulted in 171.1% and 66.62% increases in TFC for control and irradiated plants, respectively. Moreover, increasing CO2 concentration reduced the harvesting time to three and four weeks for control and irradiated plants, respectively. Enhancing CO2 to 800 µmol/mol resulted in a 193.30% (CO2 800) increase in leaf biomass compared to 400 µmol/mol and 226.34% enhancement in irradiated plants (CO2 800, G20) [800 = Ambient CO2; G20 = Plants exposed to 20 Gy] than CO2 400, G20. In addition, the CO2 800, G20 had the highest amount of flavonoid*biomass in the 4th week. The results of this study indicated that all elevated CO2 treatments had higher PN than the ambient ones. The findings showed that when CO2 level increased from 400 to 800 µmol/mol, stomatal conductance, leaf intercellular CO2 and transpiration rate had the tendency to decrease. However, water use efficiency increased in response to elevated CO2 concentration. Returning to the findings of this study, it is now possible to state that the proposed method (combined CO2 and gamma irradiation) has the potential to increase the product value by reducing the time to harvest, increasing the yield per unit area via boosting photosynthesis capacity, as well as increasing biochemicals (flavonoids) per gram DM.


Introduction
The daily intake of flavonoids in normal food, particularly fruits and vegetables, is 1-2 g. Modern physicians are increasing their use of pure flavonoids to treat many important common diseases due to their proven ability to restrain specific enzymes, to stimulate a number of hormones and neurotransmitters, and to scavenge free radicals [1]. Consumption of Centella asiatica as nutraceutical has shown to be a significant way to achieve the desired quantity of flavonoids from plant. However, flavonoids concentration in Centella asiatica is still relatively low. It is important to highlight that limited information exists on the potential and extent of enhanced production of secondary metabolites using physical elicitors, such as gamma radiation [2]. Moreover, few studies have documented the effects of ionizing radiation on photosynthesis. Therefore, it is important to examine the effects of ionizing radiation on the photosynthetic system.
In recent years, there has been an increasing interest in controlled environment (CE) plant production which reduces variation related to climate, soil, and nutrition [3][4][5], decreases contamination of samples by weeds, insects, and foreign matter [6] and enhances the standardization of secondary metabolite production [7]. As such, CE has the potential to boost the efficiency and quality of medicinal plants [8]. Additionally, a number of studies reported that CO 2 enrichment enhances the production of secondary metabolites [9] and antioxidant activity [10].
Gamma radiation can interact with atoms and molecules to build up free radical levels in cells that cause modifications in important components of plant cells. It may induce remarkable morphological changes in plant tissues, as well as various biochemical responses at the cellular level. Papers by Kim and colleagues [11] have supported the hypothesis that gamma irradiation will induce growth stimulation by altering the hormonal signaling system in plant cells or increasing cells antioxidant activity to easily overcome daily environmental stress. Besides, gamma is able to alter physiological attributes to create new mutants with improved properties that can produce higher quantity of commercially essential metabolites and develop varieties that are agriculturally and economically significant, and contain high productivity potential [12][13][14].
Recently, researchers have shown an increased interest in evaluating the product of lipid peroxidation (malondialdehyde-MDA-as an indicator of free radicals) which is able to react with amino acid residues of membrane protein and nucleic acids, reduce membrane stability, and enhance membrane permeability. Therefore, cell structure and normal physiological function are destroyed [15]. CO 2 enrichment, through provision of extra carbon to plants, is assumed to scavenge free radicals (decreasing MDA) through promotion of flavonoid biosynthesis.
The objectives of the following experiments in this study were to determine the effects of CO 2 enrichment on the growth, leaf gas exchange and total flavonoid content in irradiated and non-irradiated accessions of C.asiatica and to identify mechanisms of CO 2 enrichment and gamma irradiation effects on flavonoid concentration through MDA content of Centella asiatica.
It was hypothesised that CO 2 would increase flavonoid compounds via excess carbon, as well as alleviate detrimental effects of gamma irradiation on photosynthesis apparatus and biomass. It is expected that CO 2 and gamma irradiation would have synergistic effects to boost flavonoids in the leaves of C.asiatica.

Determination of Total Flavonoid Content and Leaf Biomass
Statistical tests revealed significant differences in flavonoid contents of Centella asiatica leaves between different growth stages and various CO 2 treatments. The total flavonoid content (TFC) in the control (9.24 ± 0.028 mg/g DW) was found to be highest after four weeks of growth, and accordingly, this stands as the best time for leaf harvesting in the chamber without any treatment. When comparing the TFC of the control plants and the irradiated ones, results demonstrate that the irradiated plants exhibited significantly greater TFC in the 5th week (16.90 ± 0.021 mg/g DW) than the control plants at their best harvesting time (4th weeks). In other words, CO 2 400, G20 (400 = Ambient CO 2 ; G20 = Plants exposed to 20 Gy) showed 82.90% higher TFC than CO 2 400 as control.
In brief, according to the results obtained in the present study, it can be concluded that plants treated with gamma radiation and high concentration of CO 2 displayed the highest total flavonoid content compared to other treatments. Moreover, the 4 th week of growth for irradiated plants exposed to high level of CO 2 was found to possess the highest flavonoid content.
Wang et al. [10] reported that strawberry fruit contains flavonoids with antioxidant properties, and CO 2 enrichment conditions enhanced the phenolic compounds, flavonol, and anthocyanin concentrations. They further noted that plants grown under CO 2 enrichment condition also had higher oxygen radical absorbance activity against [other types of oxygen] radicals in the fruit. For this reason, they concluded that atmospheric CO 2 enrichment truly enhances plant secondary metabolite. Interaction of different CO 2 concentration, gamma dosage and leaf sampling on leaf total flavonoid, leaf biomass and flavonoid * leaf biomass in plants of C.asiatica; n = 2 (400 = Ambient CO 2 ; 800 = 800 μmol/mol CO 2 concentration; G0 = Control or non-irradiated plants; G20 = Plants exposed to 20 Gy; W = Weekly leaf sampling "for instance; W4 = 4th week after planting").
Similar results were also observed in a number of vegetative tissues. For example, the growth enhancement associated with CO 2 enrichment in CE was also accompanied via a boost in the total content of flavonoids in the vegetative tissues of S. barhata and S. laterflora [16]. More specifically, the total flavonoid concentration of S. lateriflora was significantly higher than S. barbata (1144 vs. 249 µg/g DW), and S. lateriflora hada more pronounced response to CO 2 enrichment compared to S. barbata. The total flavonoid content increased by over 2.4 times at 1200 and 4.9 times at 3000 µmol/mol CO 2 [16].
Previous studies have reported that sometimes, excess carbohydrates are used to enhance the biosynthesis of secondary carbon compounds in leaves. In the study by Estiarte et al. [17], for instance, leaves of spring wheat grown at 550 ppm CO 2 exhibited 14% higher total flavanoid concentration than leaves exposed to 370 ppm CO 2 .
In general, there were significant differences among the different treatments on leaf biomass measurements. First, it was observed that increasing the concentration of CO 2 from 400 to 800 µmol/mol had significant effects on the growth and development of C.asiatica leaves, such that enhancing CO 2 to 800 µmol/mol (CO 2 800) resulted in a 193.30% increase in the leaf biomass compared to 400 µmol/mol (CO 2 400) and 226.34% enhancement than CO 2 400, G20.
Results collected after 6 weeks of growth showed that CO 2 800 had the highest leaf dry weight (151.05 ± 0.140 g/m 2 ) while CO 2 400, G20 had the lowest dry weight (41.52 ± 0.087 g/m 2 ). Furthermore, the flavonoid*leaf biomass amount [total flavonoid concentration (mg/g leaf DW) multiplied leaf biomass (g/m 2 )] was significantly different between 400 and 800 µmol/mol CO 2 concentrations. The CO 2 800, G20 in the 4th week had the highest amount of flavonoid*biomass, which was 21.8% higher than CO 2 800 in the 3rd week (as its highest level time for TFC; Figure 1).
Other studies have also reported similar results in strawberry plants. Deng and Woodward [18], for example, pointed out that after growing strawberry plants in air containing an extra 170 ppm of CO 2 , the total fresh fruit weights were 42% and 17% higher than weights exhibited by control plants grown at high and low soil nitrogen contents, respectively. Additionally, Bushway and Pritts [19] found that a two-to three-fold enhancement in the air's CO 2 content increased strawberry fruit yield by an average of 62%. Furthermore, Campbell and Young [20], Keutgen et al. [21], and Bunce [22] highlighted positive strawberry photosynthetic responses with additional 300 ppm CO 2 ranging from 9% to 197%. Finally, Desjardins et al. [23] demonstrated 118% enhancement in photosynthesis in reaction to 600 ppm increase in the air's CO 2 concentration.
To date, various methods have been developed and introduced to increase the quality and quantity of plants, and CE provides the technology for C.asiatica to respond to CO 2 enrichment by accelerating the growth rate of the plant including the leaf, producing higher biomass, and decreasing the time to harvest when CO 2 concentration is increased from 400 to 800 µmol/mol.
The response of these accessions (C.asiatica) is consistent with those reported for various food and ornamental plants over the years [5,24,25]. These growth effects are commonly connected with increased rate of leaf photosynthesis when CO2 ceases to be a restrictive factor for carbon assimilation. The experiment by Yu et al. [26] specified that exposure to UV-B alone considerably reduced dry weight and photosynthetic rate, while CO 2 enrichment alone increased dry weight and photosynthetic rate. In their study, they also observed that the dry weight and photosynthetic rate of P. subcordiformis grown under the combination of UV-B and CO 2 had no significant difference compared tothat grown under ambient UV-B and ambient CO 2 .

Leaf Gas Exchange Measurement
For this study, the net photosynthetic rate (PN), stomatal conductance (gs), intercellular CO 2 concentration (Ci), and transpiration rate (E) were determined by a portable infrared photosynthesis system LI-6400 (LI-COR, Lincoln, NE, USA) from 8:30 am to 10:30 am. During the experiment, photosynthetic photon flux density (PPFD) and leaf temperature were maintained at 1000 μmol/m 2 /s and 30 °C, respectively.
According to Jackson et al. [27], water use efficiency is defined as the ratio between the quantities of CO 2 assimilated by photosynthesis to the [quantity of] water lost through transpiration.
Based on the results gathered in this study, significant differences [in the leaf gas] were found among the four treatments.
In general, the photosynthesis rate in CO 2 800 µmol/mol had significantly the highest value compared to other treatments. All elevated CO 2 treatments had higher PN than the ambient ones. In other words, CO 2 800 and CO 2 800, G20 displayed 44.47% and 64.02% higher PN compared to CO 2 400 and CO 2 400, G20, respectively (Figures 2 and 3).
When comparing the combined effects of high level of CO 2 (800µmol/mol) and gamma irradiation treatments (20 Gy) with the control plants, it is found that there were significant differences in the photosynthesis rate. More specifically, CO 2 800, G20 had 16.95% higher PN than CO 2 400, but 23.52% lower than CO 2 800.
As shown in Figure 4, when the CO 2 level was increased from 400 to 800 µmol/mol, stomatal conductance had the tendency to decrease. The lowest gs were detected in CO 2 800, which was 56.87% lower than that for CO 2 400 as control. Moreover, combined CO 2 and gamma treatments (CO2 800, G20) resulted in 29.46% lower gs than that for CO 2 400, G20, even though CO 2 400, G20 itself had 26.89% higher gs than CO 2 800.   There were also significant differences among treatments with respect to Ci. In general, it was observed that leaf intercellular carbon dioxide declined significantly with increasing CO 2 concentration. Specifically, the Ci of CO 2 800 declined by 18.56% compared to CO 2 400 as control ( Figure 4). Moreover, combined CO 2 and gamma treatments (CO 2 800, G20) had 3.86% and 5.52% lower Ci than CO 2 400, G20 and CO 2 400, respectively, whereas its Ci was 13.8% higher than CO 2 800. Meanwhile, the highest Ci was found in CO 2 400 (289.5 ± 0.597), while the lowest was found in CO 2 800 (235.75 ± 0.790).
These findings further support the idea of increased carboxylation activity of ribulose 1,5-bisphosphate carboxylase oxygenase enzyme (Rubisco) in leaves under elevated level of carbon dioxide enhances the net photosynthesis, particularly in C 3 species. In effect, stomatal conductance will decline, which may result in less transpiration per unit leaf area. According to Morison [28], as the carbon dioxide level doubled, stomatal conductance declined by 30-40%. Nevertheless, this amount varies among species. Concomitantly, water use efficiency (WUE) will increase. The reason for this increase is mainly due to an increase in the net photosynthesis rate than the reduction of water loss through partially closed stomata. Consequently, more dry matter can be made per unit of water used [29,30].
In buckwheat leaves, the PN still declined through higher Ci value, owing to lower gs as a consequence of limited carboxylation efficiency and CO 2 assimilation, because of the excessive accumulation of free radicals in mesophyll cells by irradiation. [31]. Hence, it indicates declining of Rubisco efficiency, which means higher Ci, always can't be resulted in higher PN.
On another note, short term exposure to elevated CO 2 levels is demonstrated to promote net photosynthetic rate in C 3 plants because the existing ambient CO 2 concentration is inadequate for Rubisco [32]. An enhancement in CO 2 accessibility increases carboxylation and reduces the oxygenase activity of Rubisco (which catalyzes either the carboxylation or the oxygenation of ribulose-1 5-bisphosphate with carbon dioxide or oxygen), therefore reducing the CO 2 loss through photorespiration. Consequently, a net increase in photosynthesis takes place due to the procession of extra CO 2 [33,34]. An increase in the net photosynthesis in elevated CO 2 is predicted in spite of whether Rubisco activity or regeneration of ribulose-1, 5-bisphosphate (RubP) is restricting assimilation, and whether the light is saturating or limiting [32].
In another major study, Zobayed et al. [35] pointed out that St. John's wort plants grown under high level of CO 2 enriched condition (1,500 µmol/mol) displayed increased secondary metabolite production and net photosynthetic rates.

Determination of Lipid Oxidation (Malondialdehyde (MDA))
Changes in the MDA content of Centella asiatica accessions under different CO2 treatments are shown in Figure 6. The MDA contents of Centella asiatica accessions exposed to gamma ray and high CO2 concentration (CO 2 800, G20) were significantly decreased (65.98%) compared to those that were irradiated, but had ambient CO 2 level (CO2 400, G20). Yet, no significant difference in MDA content was detected among the non-irradiated plants exposed to different concentrations of CO 2 .
MDA is a product of lipid peroxidation and is commonly used as an indicator in stress physiology of plants. Yu et al. [26] reported that MDA content declined in P. subcordiformis grown under high levels of CO 2 , indicating that lipid peroxidation was decreased. The fact that UV-B increased MDA content revealed that the membrane was severely damaged. The combination of UV-B and CO 2 notably reduced MDA content compared to UV-B alone. Additionally, the results of this study also specified that elevated CO 2 could alleviate UV-B-induced damage to membrane.
Doubling the present atmospheric CO 2 will modify the CO 2 /O 2 ratio at Rubisco fixation site, causing 50% reduction in the ratio of photorespiration [36] and about 25%-60% enhancement in the activated state of Rubisco. Moreover, in terms of Yu et al. [26] description, the reduction in lipid peroxidation of P. subcordiformis at high concentration of CO 2 might be due to the additional carboxylation reaction of Rubisco, restricted photorespiration and less photoreduction of dioxygen. In this case, fewer electrons were transported to dioxygenduring photosynthesis and in effect, reducing the potential damage of active oxygen to membrane system. Figure 6. MDA content in different CO 2 concentration in irradiated and non-irradiated accessions of C.asiatica; n = 2 (400 = Ambient CO 2 ; 800 = 800 μmol/mol CO 2 concentration; G0 = Control; G20 = Plants irradiated to 20 Gy).

Experimental Design
All experiments involving CO 2 chamber were designed separately. For flavonoid determination, split split plot based on RCBD were used. The factors were two CO 2 concentrations (400 and 800 µmol/mol) for two hours, two irradiated and non-irradiated accessions and six leaf samplings (every week). For other parameters, the design was split plot [two CO 2 concentrations (400 and 800 µmol/mol) and two irradiated and non-irradiated accessions].

Plant Materials and Growth Chamber
The chamber model used to grow C.asiatica in this study was a PGW 36 distributed by CONVIRON. It has a microprocessor control system, in which through it, user isable to control temperature, lighting, relative humidity, carbon dioxide and other environmental conditions in the chamber. C.asiatica accessions were grown in a chamber to provide the appropriate temperature, relative humidity, and CO 2 for the irradiated and non-irradiated plants. The photoperiod and light intensity were managed in a controlled environment chamber.

Plant Harvest (Leaf Biomass)
Growth measurement and plant harvest were conducted once every week. At each harvest, the leaf biomass per plant was determined by oven-drying the leaves at 45-50 °C until it reached a constant mass.

Determination of the Total Flavonoid Content
The flavonoid contents in extracts were determined spectrophotometrically according to the method established by Lamaison and Carnat [37], which is based on the formation of a flavonoid-aluminium coloured characterized by a wavelength with a maximum absorption of 430 nm. After oven drying samples at 45-50 °C, they were kept in a −20 °C freezer. Each sample (1 g) was added to 80% methanol (20 mL) and incubated in the Orbit Shaker at 250 rmp and 50 °C for 2 hours. The samples were then filtered. Extract (1 mg) and 2% AlCl 3 methanol solution (1 mL, 2 mg AlCl 3 added to 100 mL absolute methanol) was prepared and kept at room temperature for 15 minutes. Finally, the absorbance was measured at 430 nm using a spectrophotometer. Rutin was used to create the calibration curve and the flavonoids content were expressed in mg per g of rutin equivalent (mg/g).

Estimation of Lipid Oxidation (Malondialdehyde (MDA))
The level of lipid peroxidation is stated as the content of MDA. The first method to assess the TBA-MDA complex in plant tissue was offered by Heath and Packer [38]: MDA equivalents (nmol/mL) = [(A532−A600)/155000] × 10 6 where 532 nm represents the wavelength of maximum absorption of the TBA-MDA complex, A600 is a correction for non-specific turbidity, and 155,000 is the molar extinction coefficient for MDA.
There are possible explanations for the results of the current study which indicate that the synthesis of carbon-rich secondary chemicals was restricted by the accessibility of photosynthates, and that growth processes dominated over differentiation and/or production of defence-related secondary metabolites as long as conditions were favourable for growth. When growth was more restricted than photosynthesis, then the distribution towards defence would increase.

Conclusions
Returning to the hypothesis posed at the beginning of this study, it is now possible to state that, the results of this study clearly demonstrate the potential of using CO 2 and gamma to increase the production and quality of C.asiatica. This is significant because the proposed method has the potential to increase the product value by reducing the time to harvest, increasing the yield per unit area via boosting photosynthesis capacity as well as increasing biochemicals (flavonoids) per gram DM.
Collectively, the enhancement in yield and quality provides an economic motivation to produce a consistent pharmaceutical-grade product for commercial purpose. The observations by Estiarte et al. [17], who reported higher flavonoid concentration in wheat after CO 2 enrichment, are in agreement with the results of this study. The present findings also seem to be consistent with the hypothesis that higher carbon accessibility can be invested in flavonoids.
The effect of UV-B irradiance on the growth and flavonols of Gnaphaliumluteo-album was studied by Cuadra et al. [39]. Their experiments revealed that high-irradiance treatment declined the biomass (leaf weight) and increased flavonols. This is in agreement with the results of this study, in which instead of UV, gamma irradiation was applied to create oxidative stress. In conclusion, several mechanisms can be involved in modifying C.asiatica response to CO 2 enrichment by applying gamma radiation. For example, a damaged photosynthetic apparatus under enhanced gamma or UV-B may alter the photosynthetic response to elevated CO 2 [40].
The following conclusions can be drawn from the present study, that elevated CO 2 could reduce the MDA content induced by gamma radiation, and therefore reducing the oxidative damage to C. asiatica. The most important cause for lower oxidative stress in C.asiatica grown under elevated CO 2 lies in the fact that high concentration of CO 2 possibly enhances the ratio of CO 2 /O 2 , increases the assimilation of CO 2 , declines the formation of ROS (Reactive oxygen species) as the product of O 2 acting as electron receptor, and finally reduces the formation of H 2 O 2 as the product of photorespiration, thereby reducing MDA.
This result may be explained by the fact that C.asiatica grown under high CO 2 is better in overcoming the detrimental impact of stress factor (gamma radiation) that acts via the generation of activated oxygen species. The explanation of the results for C.asiatica grown under the combination of elevated CO 2 and gamma are complex. There are, however, possible interpretations. Enhanced gamma and CO 2 concomitantly and synergistically resulted in boosting flavonoid concentration, and protecting and ameliorating the photosynthesis system. Although, CO 2 enrichment compensated the adverse effects of gamma, the plant also benefits from gamma irradiation to significantly boost flavonoids components.
These findings provide the following insights for future research on C.asiatica such as using gamma greenhouse, gamma field or ion beam supplemented with CO 2 in different types of settings such as an open top chamber, as well as applying CO 2 in a bioreactor, which remain to be done in the future.