Evaluation of α-amylase expression and analysis of phytochemical in the leaf callus tissue of Rauvolfia serpentina (Linn.) Benth. Ex Kurz exposed to Cyclodextrin

Rauvolfia serpentina (Linn.) Benth. Ex Kurz belongs to the family Apocynaceae. The objective of the present study was to establish an effective protocol for the regeneration of leaf explants from R. serpentina and to study the α-amylase expression and phytochemical profiling by gas chromatography and mass spectrometry (GC-MS). Further antibacterial activity was studied using silver, gold, and copper nanoparticles. The leaf explants were cultured on Murashige and Skoog (MS) medium containing Benzyl amino purine (BAP) (2.0 mg/L-1) and Naphthalene acetic acid (NAA) (1.0 mg/L1) induced the formation of callus and expressed α-amylase with underexposed to Cyclodextrin. Explants growing on MS medium fortified with 2,4-Dichlorophenoxy acetic acid (2,4-D) (1.0 mg/L-1), NAA (1.0 mg/L-1) with and without BAP (2.0 mg/L-1) and showed a maximum concentration of protein on the 75th day. Synthesis of a-amylase enzyme was expressed 40 days old culture and were confirmatic by western, further Silver, gold, and copper nanoparticles were synthesized using the ethyl acetate extract of callus tissue and subjected to thin-layer chromatography (TLC), which resolved 5 bands. These five bands were characterized by Fourier Transform Infrared (FTIR) Spectroscopy and screened for antimicrobial and antioxidant activities. TLC band 4 alone showed inhibitory activity against both Gramnegative and positive bacteria and potent antioxidant activity. For the first time, α-amylase was found in the callus extract by SDS-PAGE and confirmed by Western blot. The fourth band of TLC from the ethyl acetate extract as well as silver and gold nanoparticles synthesized using this extract revealed pronounced antimicrobial and antioxidant activities. GC-MS analysis revealed 26 compounds, which included mainly the phytosterols and fatty acid esters.

R. serpentine is a rich source of different varieties of chemical constituents. The root of this plant contains several alkaloids; which include ajmalicine, reserpine, serpentinine, ajmaline, ajmalimine, deserpidine, indobidine, reserpiline, rescinnamine, rescinnamidine, serpentine, and yohimbine (Anonymous, 2001;Day and De, 2011). Among the alkaloids, reserpinehas attracted worldwide attention for drug development. It is also useful in treating sedative insomnia, psychological disorders, excitement, epilepsy, traumas, anxiety, schizophrenia, insanity and in reducing blood pressure (Rai, 2004;Itoh et al., 2005; Dey and De, 2010; Azmi et al., 2013). Reserpine exerts antihypertensive property by depleting the catecholamine (Gawade and Fegade, 2012;Singh et al., 2015). Rescinnamine has the same activity like reserpine. However, it inhibits angiotensin-converting enzyme (ACE) that converts the angiotensin I, resulting in a decrease of plasma angiotensin II. Ajmaline possesses antiarrhythmic effect by blocking the sodium channel (Gawade and Fegade, 2012;Singh et al., 2017). Serpentine has antipsychotic property because it inhibits type II topoisomerase. Yohimbine is selective alpha-adrenergic antagonist in blood vessels for the treatment of erectile dysfunction (Singh et al., 2017). High concentration of phenols of R. serpentina revealed significant antidiabetic, hypolipidemic and antimicrobial properties. Flavonoids of R. serpentine help in preventing the oxidative cell damage and having anticancer, anti-inflammatory, and antioxidant properties (Harisaranraj et al., 2009;Deshmukhey et al., 2012;Kumari et al., 2013). The presence of saponins is responsible for the hemolytic activity and cholesterol binding property (Kokate, 2012).
Ethnopharmacological studies have shown the antioxidant activity with respect to superoxide anion scavenging activity, reducing power and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity by the methanolic extract of leaves of R. serpentina (Nair et al., 2012). Methanolic extract of R. serpentina rhizome also exhibited antioxidant activity as evident by the free radical scavenging activity and the increased level of glutathione peroxide, glutathione-Stransferase, glutathione reductase, superoxide dismutase, catalase, glutathione and decreased level of lipid peroxidation in CCl4-induced hepatotoxicity rat model (Gupta et al., 2015). Ethanolic extract of root was shown to possess antibacterial activity against Staphylococcus, Bacillus subtilis (Gram-positive) and Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium (Gram-negative bacteria) (Harisaranraj et al., 2009;Negi et al., 2014;Murthy and Narayanappa, 2015). Ethanolic extract of R. serpentina whole plant showed antivenom activity by neutralizing the toxic effect of Najanaja venom (Rajashree et al., 2013). Aqueous ethanolic extract of the root of R. serpentina manifested hepatoprotective activity by protecting the liver from paracetamol-induced liver toxicity in rats (Gupta et al., 2010). This extract also has reversal effect on the levels of liver glycogen, serum bilirubin, thiobarbituric acid and glutathione and the activities of superoxide dismutase, catalase, glutathione peroxide, glutathione-S-transferase, glutathione reductase and Na + K + -ATPase (Gupta et al., 2010). Azmi et al. (2012; reported the therapeutic potential of methanolic root extract in lowering the risk of atherogenic dyslipidemia, arteriosclerosis and glycosylation in alloxaneinduced diabetic mice. Ezeigbo et al. (2012) evaluated the antidiarrheal property of methanolic extract of leaves of R. serpentina in castor oil-induced diarrhea in mice.
R. serpentina alkaloids have attracted worldwide attention in International markets for their high therapeutic efficiency and drug development. Indiscriminate collection of the plant, especially roots and overexploitation for commercial purposes have threatened this species with extinction. In order to conserve this valuable endangered species, an attempt has been made to define a method for in vitro propagation of this plant species and to study the phytochemical composition by gas chromatography and mass spectrometry (GC-MS) and α-amylase expression. Further, silver, gold and copper nanoparticles were synthesized using the ethyl acetate extract of callus tissue and subjected to thin layer chromatography (TLC). The TLC bands were then characterized by Fourier Transform Infrared (FTIR) Spectroscopy and screened for antimicrobial and antioxidant activities.

Source of explants of R. serpentina
Leaf segments of R. serpentina. obtained from healthy mother plant (1-2 months old) ( Fig.1) growing in the Kalasalingam Academy of Research and Education, Krishnankoil, Tamil Nadu, India, served as explants (Figs 1a-f).

Surface sterilization of explants
The collected pieces of leaves were washed under running water for 5-10 min to clean dust particles and then by liquid detergent (Vim soap oil). Subsequently, these explants were surface-sterilized with mercuric chloride (0.1 %) for 5 min and then rinsed with distilled water five-six times.

Preparation of culture media and culture conditions
Tissue culture medium was prepared according to the method of Murashige and Skoog (1962). The medium contained 3% sucrose and solidified with 0.8% agar. The pH of the media was set to 5.8 and heat resistant growth regulators, Benzyl amino purine (BAP) and auxins like Naphthalene acetic acid (NAA) were added to the medium and then sterilized in an autoclave at 121°C under 15 psi for 15 min.
Under laminar flow cabinet, disinfected leaves were aseptically excised and placed on the media in different orientations. The cultures were maintained at 27ºC with 16 h light and 8 h dark photoperiod per day with cool white fluorescent lights at an intensity of 85 µmol m -2 s -1 . This experiment was repeated thrice. Data on callus induction and growth were recorded periodically.

Preparation of protein and SDS-PAGE gel electrophoresis
Proteins extracts were prepared by homogenizing 500 mg of callus tissue (35 days old),mature leaf and root samples separately in Tris-HCl buffer (0.1 M, pH,8.0) at 4°C. The samples were sonicated by keeping it in an ice box. Homogenates were then centrifuged at 12,000 rpm at 4°C for 10 min. Protein concentration in the supernatant was determined by the method of Bradford (1976) using BSA as a standard.
Protein profiling was carried out by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. Samples were denatured with Tris buffer (0.125 M, pH, 6.8), containing β-mercaptoethanol (5%), dithiothreitol (0.03%), glycerol (40%) and SDS (2%). The denatured protein (20 μl) was incubated in a water bath at 100°C for 3 min and loaded onto SDS-PAGE, which consisted of 12.5% separating gel and 5% stacking gel. Bromophenol blue (5 μl) was used as tracking dye. After the gel was cast, a volume of each of 15 µl protein samples from callus tissue, mature leaf and root samples were then loaded onto gels separately. A protein of known molecular weight marker standard (5 μl) (Bangalore Genei Pvt. Ltd.) was loaded in a separate lane adjacent to the sample wells. Electrophoresis was conducted at a constant current of 25 mA and a voltage of 150 V and until the bromophenol blue reached the bottom of the gel. After the run was over, the gels were carefully removed and immersed in a staining solution (0.5% Coomassie Brilliant Blue R-250) and destained in a solution containing 45% (v/v) methanol and 10% (v/v) acetic acid for 12 h. After proper destaining, the gel was documented and photographed. Molecular weight of the protein bands was determined by comparing the protein bands of molecular weight marker standards.

Western blot assay
For Western blotting, proteins that were resolved on SDS-PAGE on the basis of size were electrophoretically transferred onto nitrocelluose membranes (0.2 μm) (Millipore Corporation, USA). To block the nonspecific binding, the membranes were incubated with 5% (w/v) non-fat milk powder for 2 h. Membranes were probed with primary rabbit polyclonal anti-α-amylase (1:2000) antibody overnight. The membranes were then extensively washed and incubated with the horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The bands were developed using ECL kit (Millipore, Bangalore, India). Protein expression levels were visualized with the Image Lab software (Bio-Rad, USA). Image densities of specific bands for α-amylase were normalized with the density of β-actin.

Preparation of callus tissue extracts
Thirty five days old mature callus tissue was extracted with ethyl acetate for 8 h and concentrated using rotary evaporator and stored in a desicator until use.

Synthesis and characterization of silver, gold and copper nanoparticles using callus tissue
Silver, gold and copper nanoparticles were synthesized by mixing 10 ml of ethyl acetate extract of callus tissue with 100 ml of aqueous solutions of silver, gold and copper nitrate separately with constant stirring at room temperature. The mixtures were heated at 60°C and then cooled to room temperature and kept in dark for 24 h. The color change of the mixture was recorded visually. The surface morphologies and size of the silver, gold and copper nanoparticles were examined using Scanning Electron Microscopy (JSM-6360, JEOL), attached with Energy dispersive X-ray (EDX) diffractometer (Carl Zeiss, Germany).

TLC method
TLC is a method for separating the compounds from the mixture and determining the identity and purity of the compounds. In the present study, an aliquot of ethyl acetate extract of callus tissue was spotted on TLC silica gel plates (10 x 15 cm). The plates were developed using hexane and ethyl acetate (8:2) as the mobile phase. After completion of the run, the plates were taken out from the development chamber, air dried and visualized under visible and UV light (240 and 300 nm). In the present study, the chromatogram revealed five distinct bands. The separated bands were marked and their retention factor (Rf) values were calculated and recorded. The chromatogram was then photographed.
The five bands of TLC chromatogram were scratched off separately, dissolved in alcohol, filtered, concentrated and then used for the antioxidant and antibacterial assays.

Antibacterial activity
The antibacterial activity was checked by TLC bioautographic method using Gram negative [Escherichia coli (MTCC 1652) and B. licheniformis (MTCC 73537)] and Gram positive [Staphylococcus aureus (MTCC 96) and Pseudomonas aeroginasa (MTCC 2453)] bacteria (NCCLS, 1993). All bacterial strains were provided from the microbiology laboratory of the Meenakshi Mission Hospital. All Bacterial strains were subcultured in nutrient agar broth for 24 h prior to testing.
In the present study, TLC chromatogram showed five bands. Each of the five bands of TLC was scratched off separately, mixed with 5 ml of absolute ethanol, allowed to stand for 10 min and then filtered with Whatman No. 1 filter paper and collected in glass vials. The recovered concentrates of each band were then tested for antibacterial activity by agar diffusion method.
About 0.1 ml of inoculam (1.5 x 10 8 /ml) of each bacterial strain was streaked out on molten Mueller Hinton agar plates with a sterile cotton swab. Wells of 7 mm diameter were made by scooping out agar with a sterile cork borer. The recovered concentrates of each of the five TLC bands were dissolved in 10% DMSO separately and loaded into the wells (200 µg/ well). A control well was added with 10% DMSO alone and served as negative control, while amphicilin (20 µg) was used as the positive control. Tests were carried out in triplicates and the plates were observed for the zone of inhibition and the diameter of the same was measured in cm.
Further, antibacterial activity of three nanoparticles (gold, copper and silver) prepared using ethyl acetate extract of callus were also checked against two bacterial strains E.coli (Gram negative) and S. aureus (Gram positive)bacteria.

Determination of total antioxidant activity
Like antimicrobial activity, antioxidant activity was determined by TLC bioautographic method. Each of the five bands of TLC chromatogram was scratched off, mixed with absolute ethanol, filtered and concentrated. The recovered solutions of each band were then tested for total antioxidant activity (Prieto et al., 1999).
Total antioxidant capacity was assessed by phosphomolybdenum method. The assay is based on the reduction of molybdenum (VI) to green phosphate/molybdenum (V) complex at acidic pH by the sample analyte.
An aliquot from the recovered solutions (0.3 ml) of each TLC band was mixed with 1 ml of reagent solution containing sodium phosphate (28 mM), sulphuric acid (0.6 M) and ammonium molybdate (4 mM), incubated at 95°C for 90 min and then cooled to room temperature. The intensity of green color developed was read at 695 nm using a double beam spectrophotometer (UV-160 A, Shimadzu Corporation, Kyoto, Japan) against a blank. The total antioxidant activity is expressed as the number of gram equivalent of ascorbic acid.

FTIR analysis
FTIR spectral analysis was performed by TLC bioautographic method using the recovered concentrates of five TLC bands obtained by using ethyl acetate extracts of 35 day old callus tissue. FTIR spectral analysis was performed in FTIR instrument (IRTRACER-100, Shimadzu, Japan) in the region of 4000 cm −1 to 500 cm −1 with PC based software and data processing. As mentioned earlier, each band of TLC was removed separately, mixed with absolute ethanol, filtered and concentrated. The recovered concentrates of each band was then encapsulated using KBr (100 mg) pellets in order to prepare translucent sample discs by applying pressure for FTIR analysis.

Phytochemical screening of ethyl acetate extract of callus tissue by GC-MS
The phytochemical screening was carried out in the ethyl acetate extract of 35 day old callus tissue by GC-MS technique. GC-MS analysis was carried out in an Agilent gas chromatography N6890 fitted with a HP-5MS fused silica column (5% phenyl methyl polysiloxane 30 m x 0.25 mm, film thickness 0.25 μm), interfaced with an Agilent 5975C VLMSD with triple axis mass detector. One microlitre of the sample was injected to the injected port. The oven temperature was raised from 40°C to 220ºC at a rate of 6˚C/min. Helium was used as the carrier gas with a flow rate of 0.5 ml/min. Split ratio was 1:10, whereas split flow of 10 ml/min-1 mass range was 50 to 500. The sample was vaporized and then the various components of the sample was separated and analyzed. The MS was taken at 70 eV of ionization energy. GC-MS analysis produces a specific spectral peak for each component that get separated from the sample. GC-MS chromatogram was recorded on a chart electronically. The peak was measured from the base to the tip of the peak. The time elapsed between injection and elution is called the "retention time". Retention indices (RI) of the compounds were determined by matching the spectra with reference spectra.

Identification of components
In the MS Program, National Institute Standard and Technology (NIST), Version 14.0, Wiley 8.0 library database of NIST having more than 62000 patterns was used for identifying the chemical components. The unknown phytochemicals were identified by comparing their mass spectra with the spectrum of known compounds stored in the NIST library.

Statistical analysis
Data were analysed by one-way analysis of variance (ANOVA). All the measurements are expressed as mean ± standard errors of means. A p value of < 0.05 was considered to be significant.

Induction of callus
The current study provided a protocol for large scale callus propagation of R. serpentina leaf explants. In the present study, during callus initiation, the explants did not show any leaching or browning of tissues. This indicates that the MS basal medium was the most effective for callusing of leaf explants.

Studies on a-amylase expression in callus tissue, mature leaves and roots by SDS-PAGE
SDS-PAGE is the most widely used analytical method to resolve components of a protein mixture. In the present study, the total protein was estimated from mature cream colored callus, mature leaves, and roots. Protein profiling of mature leaves, root and leaf callus by SDS-PAGE resolved around 16 bands ranging from 2 to 240 KDa (Fig.2a). Protein profiles further showed variability in the number of bands, band pattern and band intensity. Out of 16 protein bands, molecular weights 5 to 240 kDa have shown the same pattern of protein banding in leaf callus, mature leaves and root samples. The callus tissue revealed 4 bands at 50, 40, 18, and 13 kDa. The root sample also showed similar protein bands with varying intensities. However, all the four bands were found to be absent in mature leaf samples. From these observations, it is inferred that callus showed the highest number of protein bands followed by root. 50 kDa protein found predominantly in both callus and root confirms the α-amylase enzyme expression by comparison with reference sample. This was further confirmed by Western blotting (Fig. 2b).  Table 1). The callus grown on medium containing only NAA (0.1 mg/L -1 ) and 2,4-D (2.0 mg/L -1 ) showed a steady increase in protein content on 3 rd 24 th , 30 th days. Thereafter, the protein content declined. Whereas the protein content observed in the callus grown on medium fortified with NAA (0.1 mg/L -1 ) + 2,4-D (2.0 mg/L -1 ) and BAP (2.0 mg/L -1 ) showed maximal increase on the 30 th day and this level was maintained in 40 and 75 day old callus ( Table 1). The observed increase in the protein levels on, 3 rd , 24 th and 30 th days may be attributed to the mitotic activity occurring during the exponential and linear growth phases. This reduction in protein levels after 30 days may possibly be occurred due to the differentiation phase.

Characterisation of silver and gold nanoparticles
SEM analysis revealed that silver nanoparticles synthesized through green chemistry are well dispersed and spherical in shape (Figs 4a and b). Fig. 5 shows the SEM images of the gold nanoparticles. SEM images showed that most of the gold nanoparticles are highly homogenous and predominately spherical in shape having smooth surface (Fig. 5a). EDX analysis revealed the presence of silver and gold elements, confirming the successful synthesis of silver and gold nanoparticles (Figs 4c and 5c).

Antibacterial activity using agar well diffusion method
Antibacterial activity was evaluated using the recovered concentrates of five TLC bands eluted using the ethyl acetate extracts of 35 day old callus tissue. Antibacterial activity was tested against Gram negative E. coli and P. aeroginosa and Gram positive bacteria S. aureus and B. licheniformis by agar well diffusion method. If the sample examined had antimicrobial activity, a clear zone would be formed on the surface of the agar, representing an inhibition of bacterial growth.

Figure 3 TLC chromatogram showing bands under white light and UV light
In the present study, TLC chromatogram revealed five bands (Fig.3) interestingly, of the five bands of TLC tested, 4 th band or fragment alone has shown pronounced antibacterial activity with maximum inhibition zone of diameter 1.7 cm against gram positive bacteria and the lowest inhibition zone of diameter 1.2 cm in gram negative bacteria. The other four concentrates of TLC bands, (TLC band-1, TLC band-2, TLC band-3 and TLC band-5) were found to be ineffective against all the tested bacteria ( Fig.6) Nevertheless, the positive effect observed with the concentrate of TLC band-4 indicates that the R. serpentina callus synthesizes compounds responsible for antibacterial activity. This is in fair correlation with a number of earlier studies, which have shown good antibacterial activity in the leaf, shoot and root extracts of R. serpentine (Negi et al., 2014; . Murthy and Narayanappa, 2015).

Antioxidant activity
The total antioxidant activity of recovered concentrates of five TLC bands obtained using ethyl acetate extracts of 35 day old callus tissue was evaluated by comparing reference compound, ascorbic acid. Of the 5 TLC compounds tested, 4 th band expressed the highest antioxidant activity and was more than the ascorbic acid (Fig. 8).

FTIR analysis using TLC products
In the present study, FTIR spectra was obtained from the recovered concentrates of five TLC bands obtained by using ethyl acetate extracts of 35 day old callus tissue. TLC band pattern in normal and UV light are shown in Fig.3. The functional groups were identified by comparing the peak values in the IR spectra with that of the reference compounds. The FTIR spectral data of both control (silica gel alone) and the 5 bands obtained on the TLC using ethyl acetate extract of R. serpentina callus tissue are presented in (Fig. 9a and 9b). The samples were analyzed in the spectral region of 500 to 4000 cm −1. All the five bands of TLC exhibited a characteristic absorption maxima at 2386 cm −1 , indicating the presence of C-H stretching. Another characteristic absorption maxima at 1095.57 cm −1 , indicating the presence of C-O) for a hydroxyl (-OH), which was observed in all the TLC bands, except band-3. TLC bands-3 and 5 revealed another characteristic peak at 1540-1560 cm -1 , which is indicative of C=O aromatic stretch. Besides, the five TLC bands showed the absorption maxima at 3000-4000 cm -1 , indicating the presence of hydroxyl groups, which includes H-bonded OH stretch, polymeric OH stretch, dimeric OH stretch and nonbonded hydroxyl group of primary, secondary, tertiary alcohol and phenol. The absorption maxima at 2882 cm -1 and 2380 cm -1 (for C-H stretching), at 1641 cm -1 (C=C stretching), 974 cm -1 (C-H bending of aromatic hydrocarbons) and 798 cm -1 (aromatic carbons) ( Table 2). Phytochemical screening of callus is required to identify the nature of bioactive components in order to find novel therapeutic agents with better efficacy. The spectral peaks in the chromatogram were compared with the spectrum of known compounds stored in the NIST library. The identified compounds, their retention time (RT), molecular weight, molecular formulae, and percentage composition (% area) are given in Table 3. A distinct chromatogram of callus tissue extract of R. serpentina is shown in Figure 10. The structure of individual components is illustrated in (Table 3). In the present study, GC-MS chromatogram shows the presence of 26 different peaks which confirm the presence of 26 compounds with their respective RT (Fig. 10)   Octasiloxane, 1,1,3, 3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl-C16H50O7Si8 579. 2 7.78 The biological activities of the phytoconstituents are listed in (Table 4). Based on the spectral data, it was found that the extract of callus of R. serpentine contained a large number of bioactive compounds including phytosterols and fatty acids.
Most of these phytoconstituents are reported to possess pharmacological potential. The presence of various bioactive compounds justifies the propagation and use of this callus tissue for phytopharmaceutical purposes.

Conclusion
Taken together, the present study provided a rapid protocol for callus initiation and growth from leaf explants of R. serpentina exposed to cyclodextrin in MS medium. For the first time, α-amylase was found in the callus extract by SDS-PAGE and confirmed by Western blot. The fourth band of TLC from the ethyl acetate extract as well as silver and gold nanoparticles synthesized using this extract revealed pronounced antimicrobial and antioxidant activities. GC-MS analysis revealed 26 compounds, which included mainly the phytosterols and fatty acid esters. The presence of these compounds in the callus tissue of R. serpentina indicates that they are promising candidates for therapeutic use and food supplementation of nutraceuticals.