Estimation of Metabolites and Antioxidant Activity in UV-C Treated Callus Cultures of Bacopa monnieri (Linn.) Pennell
1Department of Biotechnology, Vels University, VISTAS, India
2Department of Botany, Ramakrishna Mission Vivekananda College, India
*Correspondence to: Kathiravan G, E-mail: email@example.com
Citation: Meenashree B, Kathiravan G, Manickamoorthi N (2018) Estimation of Metabolites and Antioxidant Activity in UV-C Treated Callus Cultures of Bacopa monnieri (Linn.) Pennell. SCIOL Biotechnol 2018;1:9-14.
Various techniques have been used to increase the production of metabolites by plants. In the current study, the calli raised from the leaf explants of Bacopa monnieri were irradiated with UV-C light (254 nm) for 2, 3, 4 and 5 minutes. The phytochemicals were quantitatively analyzed to examine the effects of UV-C irradiation on the metabolite production. Protein was estimated by Lowry's method and it showed that on an average, the calli treated with UV-C for 4 minutes contained maximum protein content. The total phenolic content was found to be highest in 2 min followed by control, 4 min, 3 min and 5 min. The antioxidant activity was estimated using DPPH radical scavenging method. 4 min treated callus was found to show highest antioxidant activity. This study shows that UV-C irradiation has significant impact on the secretion of metabolites with antioxidant potential in Bacopa monnieri.
UV-C radiation, Callus, Phytochemicals, DPPH assay, Protein estimation
Bacopa monnieri, commonly known as water hyssop is widely being used as a memory enhancer. Apart from boosting the brain activities, the plant reportedly protects the brain against neurodegenerative disorders like Parkinson's and Alzheimer's . Oxidative stress is known to contribute to the aging process and is linked to neurodegenerative disorders. Results obtained from in vitro and in vivo studies have shown that the extract of B. monnieri decreases the free radical accumulation in the brain by enhancing the defense against oxidative stress . Phenolic compounds are known to alter the oxidants to non-radical end products and thereby providing protection. Phenolic compounds like chlorogenic acid, caffeic acid were reported in the shoot extracts of B. monnieri . The secondary metabolites which impart these health benefits are getting overexploited during drug preparation. Many strategies like exposure to extreme temperatures, wounding, water starvation and radiations have been used to produce plants with enhanced phytochemical contents . A report suggests that by exposing the plant materials to ionic and non-ionic radiations, the phytochemical production can be enhanced to great extend . γ-irradiation has been shown to enhance the plant's antioxidant activity and total phenolic contents [6,7].
UV-C irradiation has been shown to have some control over the expression of certain genes which are involved in the growth of plants and their secondary metabolism . Earlier, grape calli were treated with UV-C irradiation and showed increased accumulation of resveratrols and piceids . UV light at the range of 260-270 nm showed a positive effect on the formation of resveratrol and isopentenylresveratrol in the callus of peanut. UV-C irradiation was shown to reduce post-harvest diseases by inhibiting spore germination and enhance the antioxidant activities in Yali pear . In the case of tomato fruits, UV-C was shown to induce resistance to Rhizopus soft . When compared to UV-A it was found that UV-B and UV-C gave increased concentration of phytochemicals in pigeon pea leaves . UV-B treated Ginko biloba callus was found to show increased nitric oxide production, nitric oxide synthase (NOS) activity and thereby increased flavonoid accumulation. Vitis vinifera callus, when exposed to UV-C irradiation gave greater accumulation of resveratrol, a stilbene compound .
These reports suggest that UV-C irradiation might have the potential to improve and enhance the capacity of the plants to produce metabolites. This study was conducted to investigate the effects of UV-C irradiation on the phytochemical content of the callus culture of Bacopa monnieri, with an attempt to evaluate the role of UV-C rays in crop improvement. This is the first report to present the effect of UV-C irradiation on the callus of brahmi.
Materials and Methods
Modified MS media  containing two different hormone concentrations; 1.5 mg/L NAA + 0.5 mg/L BAP and 1.5 mg/L 2,4-D + 0.5 mg/L BAP individually, was used to initiate the callus culture. Leaf explants were surface sterilized using 0.1% w/v mercuric chloride for 4 min followed by rinsing in sterile distilled water to remove the trace mercuric chloride. The sterile explants were inoculated on modified MS media. The tubes were incubated at 25 ℃ ± 2 ℃ for 30 days in darkness to prevent browning of the callus.
Philips low pressure UV lamp (11 W) with wavelength of 254 nm was used as a source of UV-C rays to irradiate the calli. The calli were irradiated for 2, 3, 4 and 5 min individually at a distance of 30 cm and all the treatments were performed in triplicates. After the treatment, the calli were immediately transferred to modified solid and liquid MS media along with 2 mg/L BAP + 0.1 mg/L NAA for multiplication of the calli. In the case of liquid MS media, the callus was inoculated on top of a sterile whatman filter paper kept immersed on the surface of 20 mL of liquid media in a sterile boiling tube. The treated calli were allowed to multiply in darkness at 25 ℃ ± 2 ℃ for two weeks.
Total protein estimation
The concentration of protein was determined by the Lowry's method. 0.1 g of dried callus was boiled in 10 mL of sterile distilled water for 5 min. The boiled extract was cooled and the filtrate was used for protein estimation. Bovine Serum Albumin (0.1 mg mL-1) was used as a standard. Untreated callus was used as the control. Different volumes of the standard and callus extract (0.2, 0.4, 0.6 and 0.8) were taken separately in the test tubes. The volume was made up to 1 mL using distilled water. 5 mL of Biuret reagent was added to all the tubes and incubated for 10 min. 0.5 mL of diluted Folin-ciocalteu reagent was added and incubated at room temperature for 10 min followed by further incubation at 25 ℃ for 30 min. The blue color developed was measured at 660 nm in UV/V is spectrophotometer. A standard graph of BSA was plotted and the amount of protein in the sample was calculated from the linear equation. It was expressed as µg BSA equivalents per g of sample (µgg-1) .
Estimation of phenolic content
Phenols were quantified by Folin and Ciocalteu's method. Callus extract was prepared by shaking in methanol at 1 mg/mL concentration for 2 hrs. Then it was filtered and 0.250 mL of the respective callus extract was mixed with 1.25 mL of 10% v/v Folin-ciocalteu reagent and 1 mL of Sodium Carbonate. The mixture was incubated for 30 min in darkness. Methanol served as the blank. The absorbance was measured at 760 nm in UV/V is spectrophotometer. The total phenol content was obtained from the linear equation derived from standard and was expressed as mg gallic acid equivalence (GAE) per gram of callus on dried basis .
DPPH free radical scavenging assay
The callus extract was prepared by heating 1 g of callus in 25 mL of distilled water at 40 ℃ for 10 min. 0.2 and 0.8 mL of callus extract in 0.8 and 0.2 mL of methanol respectively, was added to 3 mL of 0.004% w/v DPPH in methanol. The mixture was incubated in darkness for 30 minutes. The absorbance was read at 517 nm. Ascorbic acid at a concentration of 2 and 8 mg/mL of methanol was used as a standard. 1 mL of methanol and 1 mL of DPPH served as a control. Methanol served as blank. The percentage inhibition of DPPH free radical scavenging activity was calculated by using the following equation:
All the experiments were carried out in triplicates. The mean ± standard error of the obtained result was calculated using SPSS software.
Callus initiation and multiplication
When 1.5 mg/L 2,4-D + 0.5 mg/L BAP combination was used for initiation of callus culture, no growth was observed due to browning of the explant. In the case of media containing 1.5 mg/L NAA + 0.5 mg/L BAP, pale yellow colored well-growing callus was obtained, as shown in Figure 1. After the UV-C irradiation, when the calli were transferred to liquid media extensive browning was observed which ultimately led to the decline of growth. Solid media was found to be appropriate and it gave excellent growth.
Figure 1: Callus cultures of Bacopa monnieri A) Initiation of callus; B) Multiplication of 4 min UV-C irradiated callus in solid MS media containing NAA + BAP. View Figure 1
Total protein estimation
Standard curve was prepared using the optical density (OD) values of Bovine serum albumin (BSA) obtained and the linear equation was derived from it. The concentration of protein present in the treated and control calli was calculated from the derived formula, and depicted in Table 1. Linear equation:
y = 0.005x + 0.23
Where x is the OD value and y is the protein concentration
Among the five samples, 4 min UV-C irradiated callus was found to have highest protein content on an average followed by 5 min, 3 min, control and 2 min.
The protein concentration of each sample is given as the mean value ± standard error.
In case of 0.2 mL sample in 0.8 mL water dilution, 5 min treated callus was found to have the maximum concentration of protein followed by control, 4 min, 3 min and 2 min. This shows that as the time of exposure increases the concentration also increases. In case of 0.4 mL in 0.6 mL dilution, 4 min showed highest concentration followed by 5 min, 3 min, control and 2 min. Here, the control was found to have less protein content when compared to the treated ones. For 0.6 mL in 0.4 mL dilution, 4 min was highest followed by 5 min, 3 min, control and 2 min. Finally, for 0.8 mL in 0.2 mL dilution the decreasing order of protein concentration was found to be 4 min, control, 3 min, 5 min and 2 min. On an average, 4 min treated callus gave best results.
The absorbance values of the gallic acid standard were plotted and the linear equation was derived. The absorbance values of the samples were substituted in the place of x and the phenol concentration was calculated. Linear equation:
y = 0.009x + 0.21
Where x is the OD value and y is the phenol concentration
The phenol content was found to be highest in 5 min UV-C irradiated callus followed by control, 2 min, 4 min and 3 min.
Even though 5 min UV-C irradiated callus showed highest concentration of phenol, the other treated samples showed a drastic decrease in the phenol content. Only the 5 mins treated callus was found to show promising results, it had phenolic content higher than the control. In case of 2 min, 3 min and 4 min treated calli, the phenol content was extremely low. The obtained results are graphically represented in Figure 2.
Figure 2: Phenol concentration in UV-C treated and control calli. The phenol content in the methanol extract of 2, 3, 4, 5 min UV-C irradiated calli and control. View Figure 2
DPPH free radical scavenging assay
The antioxidant potential of the treated and control calli was studied by evaluating the radical scavenging activity. The percentage of inhibition was found to be highest in case of 4 min treated callus followed by 3 min, 5 min, 2 min and control.
From the graph (Figure 3), it is evident that the antioxidant activity is extremely low in case of untreated callus when compared to the treated calli. As the time period of UV-C irradiation increased, the antioxidant potential also increased to some extent. The lines depict that 4 min and 3 min treated calli show greater antioxidant activities when compared to the untreated callus. 5 min and 2 min treated calli also showed commendable increase.
Figure 3: DPPH radical scavenging activity of test samples and standard. The antioxidant activity in 2, 3, 4, 5 min UV-C irradiated calli, control calli and standard ascorbic acid. View Figure 3
The calli and nodal segments of Bacopa monnieri have previously been exposed to gamma irradiation in order to induce variations and increase bacoside accumulation . Another study done by Nayak, et al.  showed that gamma radiated leaves produced more bacoside-A, whereas ethyl methanesulfonate treated leaves did not show any major increase. Escandon, et al.  produced two tetraploid plants with significant changes in the color and size of leaves, by treating the nodal segments of brahmi in colchicine. The UV-C treatment was found to increase the secondary metabolites' accumulation in the callus of Vitis vinifera . Likewise, many studies have been conducted on the callus of grape cultivars. Ashari and Pour  showed that callus cultures of Iranian grapes treated with UV rays showed enhanced production of resveratrol. Khatami and Ghanati  showed an increase in the flavonoids, anthocyanins and tannins production in the UV-C and UV-B treated calli of Malva neglecta.
Till date there hasn't been any study conducted to evaluate the effect of UV irradiation on in vitro cultures of Bacopa monnieri. This study is the first of its kind to report how UV-C irradiation affects the antioxidant potential and metabolite production by the callus. The results obtained from the radical scavenging assay clearly show that the treated calli are superior to the untreated ones. There was a significant increase in the total protein content in some of the treated calli. However, there was a decrease in the concentration of phenol post UV-C irradiation. The antioxidant potential was greatly enhanced in the treated calli and Figure 3 clearly depicts the significant increase when compared to the control. The results suggest that UV-C irradiation is a useful technique which can be used to enhance the accumulation of phytochemicals in the callus cultures of Bacopa monnieri.
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