A Comparison of Chemical Profiles of Callus, Plant and Agarwood Extracts of Gyrinops walla using Thin Layer Chromatography

A Comparison of Chemical Profiles of Callus, Plant and Agarwood Extracts of Gyrinops walla ABSTRACT Agarwood produced by Gyrinops walla Gaertner, an endemic plant species of Sri Lanka, arguably the costliest wood, is used as a major constituent in cosmetic industries. Application of plant cell culture technique serves as the best alternative for the production of fragrance compounds of G. walla while ensuring the sustainability of the species. Using the advantages of


INTRODUCTION
Gyrinops walla is an endemic plant species to Sri Lanka, which belongs to family Thymalaeceae. The agarwood is the most valuable and exalted perfumery raw material obtained from the infected, resin-impregnated wood of G. walla (Subasinghe and Hettiarachchi, 2013). Increasing global demand for agarwood for agar oil as the base of world's most expensive perfumes, cause extensive exploitation of natural population of G. walla from tropical rain forest areas in Sri Lanka. Therefore, it is necessary to explore alternative and efficient techniques for fragrance compound production to meet the demand, while ensuring the sustainability of the species, since fragrance compound production of G. walla could become one of the major sources of foreign income to the country. Though artificial induction of agarwood compounds is a possible method, the formation of resinous portion inside the wood takes a considerably long time. Therefore, in vitro plant cell culture is a suitable and feasible method, which could be commercially exploited for the production of quality fragrance compounds (Shu et al., 2005;Okudera and Ito, 2009).
Callus develops in response to chemical or physical lesions under determinate hormonal conditions (Jeyaraman et al., 2013). The exogenous supply of plant growth regulators is necessary in calogenesis (Nurazah et al., 2009). This necessity refers to the type and concentration of plant growth regulators mainly auxins and cytokinins, genotype of the donor plant, and the endogenous content of hormones (Lima et al., 2008). Indeed, plant cell culture technique is found to have potentials in supplementing traditional agriculture in the industrial production of desirable plant metabolites in flavour and fragrance industries (Vanisree et al., 2004).
Though there are many sophisticated techniques to analyse the chemical constituents, thin layer chromatography (TLC) is the simplest and the most versatile technique, which has potential to detect even microgram quantities of particular chemicals within a short period. These advantages bring about the possibility of using this technique in screening the chemicals present in plant extracts (Itankar et al., 2015). The TLC fingerprint with a visible pattern of bands provides fundamental data of compounds present in a sample and can be defined as a set of characteristic chromatographic signals, whose comparison leads to unambiguous sample recognition. TLC also facilitates to analyse mixtures by separating the compounds and identifying them. Furthermore, TLC profiles can be used to construct the fingerprints for specific plant species for its identification.
Scientific studies on agarwood of G. walla have been mostly focused on chemical profiling of the wood and oils. Intensive work therefore needs to be undertaken to compare the chemical profiles of callus with plant and agarwood samples of G. walla. Hence, the present study was conducted for the identification of feasibility in producing chemical profiles similar to plant and agarwood of G. walla through cell culture technique in order to ensure the sustainable utilization of this plant.

Development of callus culture on different culture media
Immature leaf explants were used to establish callus cultures and surface sterilization was performed as in the method described previously by Selvaskanthan et al. (2017). All tissue culture techniques were conducted under aseptic conditions in a laminar flow cabinet (Labgard Class II, Type A/B3). Explants were cultured on modified Murashige and Skooge (MS) medium (Murashige and Skooge, 1962) that contained half level of ammonium nitrate (NH4NO3) supplemented with 3 percent sucrose (w/v), different concentrations and combinations of cytokinins such as 6-Benzylaminopurine (BAP), kinetin and auxins such as 2,4-Dichlorophenoxy acetic acid (2,4-D), 1-Naphthalene acetic acid (NAA) and Indole-3butyric acid (IBA). Medium pH was adjusted to 5.8 with 1.0 mol/L HCl or NaOH prior to solidifying with 0.2% (w/v) Phytagel (Sigma, UK) and autoclaved at 120 °C, 15 psi for 20 minutes. Callus lines were further multiplied by sub-culturing at 6 week-intervals as decided from growth curves. Based on percentage callus induction and growth kinetics 10 callus lines grown on different media (Table 1) were selected for the detection of chemicals by TLC.

Preparation of extracts
Freeze dried calli were crushed to make a fine powder by using a mortar and a pestle. Plant samples and agarwood were ground using a heavy-duty grinder (WARING, HGBTWTS3, Waring Commercial, Torrington, USA). They were then subjected to ultrasound assisted solvent extraction by adding 100 ml of hexane and placing in an ultrasonic cleaner (ROCKER ultrasonic cleaner, model-soner 206H) for 30 minutes of extraction period before filtering through Whatman number 1 filter paper. The solvent was evaporated to dryness using rotary evaporator (Heidolph, Laborota 4000), keeping the temperature below 40 o C, at the speed of 90 rpm to obtain viscous semi-dried crude extracts. This process was repeated thrice. The residual material was sequentially extracted with ethyl acetate and methanol and evaporated to dryness by rotary evaporator. These crude extracts were subjected to TLC analysis.

Selection of the best extraction and eluting solvents for silica gel thin layer chromatography
To choose the best mobile phase for best elution, two callus lines with good callus induction percentage and specific growth rate were used. Extracts were spotted using capillary tubes on TLC silica plates pre-coated on aluminium foil (Merck, 105554, TLC Silica gel 60 F) and the solvents were allowed to evaporate completely. The spotted plates were placed in a chromatographic solvent chamber which contained 5 different eluting solvent systems viz: i. 15%, ii. 10%, iii. 5% Methanol (MeOH) in Chloroform (CHCl3), iv. 100% CHCl3 and v. 7:3:1 CHCl3: MeOH: H2O, to select the suitable mobile phase. The TLC plate was propped vertically in solvent chamber and stand allowing sufficient time for elution. When the solvent front has nearly reached the top of the stationary phase, the plate was removed from the chamber and the developed TLC plates were air-dried. UV monitoring at appropriate wavelength (254 nm) was performed for a comprehensive detection of various compounds present in the crude extract using a UV lamp (VILBER LOURMART CN-15-LC, 230 V-50/60 Hze).
Based on numbers and intensity of TLC spots, best solvent extract was chosen for further studies.

Detection of the effect of different plant growth regulators on the product synthesis by TLC
Ethyl acetate extracts of all 10 callus lines (Table 1) were evaluated to study the effects of different plant growth regulators and nitrogen level of media on product synthesis by TLC. In addition, extracts from plant stem cuts, leaves and bark were spotted on a TLC plate, kept under best solvent system and observed under 254 nm UV light. Best extracts of callus lines and plant materials were chosen and compared with the extract of agarwood under 254 nm UV light.

Selection of the best extraction and eluting solvents for silica gel thin layer chromatography
Several factors determine the efficiency of a chromatographic separation. The adsorbent should show a maximum selectivity towards the substances to be separated so that the differences in rate of elution will be large. Silica gel is the most common adsorbent used for routine TLC of organic compounds. The eluting solvent should also show good selectivity in its ability to desorb the compounds that are being separated. The solubility of different compounds in the eluting solvent plays an important role in determining how fast they move up the TLC plate. However, a more important property of the solvent is its ability to be adsorbed on the adsorbent. Based on the extent of affinity of the solvent for the adsorbent, different compounds move at different rates, resulting in separation.
Among the three solvents (hexane, ethyl acetate and methanol) used in this study for extraction, ethyl acetate extracted a higher number of phytochemicals present in callus of G. walla. (Plate 1b). Therefore, ethyl acetate was selected as the best solvent for TLC. The ethyl acetate extracts of two callus lines C6 and C10 (chosen based on higher growth rates and fastest cell doubling) were run on different eluting solvent systems. The highest number of TLC spots was observed when the ethyl acetate extracts were eluted on 15% methanol: chloroform (Plate 2 e) compared to other eluting solvents (Plate 2 a-d). Therefore, the eluting solvent system of 15% methanol: chloroform was selected as the suitable mobile phase based on the elution of TLC spots (Plate 2 e).

Detection of the effect of different plant growth regulators on the product synthesis by TLC
TLC fingerprint with a visible pattern of bands provides fundamental data and is typically used to determine the number of components in a mixture and to identity them. The compounds are identified by comparing the Rf of the compound with the Rf of a known compound, when both compounds are run on the same TLC plate (Reich and Schibli, 2006).
The effects of varying concentrations and combinations of plant growth regulators and reduced NH4NO3 contained MS medium on callus morphology and growth are shown in Plate 3. Results of the experiment done to study the influence of different plant growth regulators in culture media on product synthesis, using the 15% methanol: chloroform solvent system, showed that there was a strong influence of plant growth regulators on product synthesis (Plate 4).  (Table I). Furthermore, when the media contained kinetin, thickness of these 3 spots (C5, C6, C7) were also increased compared to the media consisted with 2, 4-D (C1, C2, C3, C4).

Plate 4: TLC fingerprint profile of ethyl acetate extracts of callus lines developed on MS (a) and modified MS medium (b) supplemented with different combinations and concentrations of plant growth regulators under 15% methanol: chloroform.
Note The results of the present study indicated that by reducing the NH4NO3 concentration to half, product synthesis of G. walla callus cultures can be enhanced. Ochoa-Villarreal et al. (2016) and Bhojwani and Dantu (2013) have reported that modification of the carbohydrate: nitrogen ratio is an effective technique to enhance the production of secondary metabolites. Ratnadewi (2016) mentioned that the nitrogen concentration of the culture medium influence the synthesis of alkaloids.
Research by Liu et al. (2013) revealed that in Anisodusa cutangulus hairy root culture, alkaloid yields were inhibited at low or high total nitrogen concentrations and the most favorable concentration of nitrogen gave the highest tropane alkaloid yield. Sujanya et al. (2008) reported that reduction in nitrogen source in cell suspension cultures of neem enhanced production of extracellular azadirachtin by 1.5-fold.
When ethyl acetate extracts of all the callus lines were cultured on different media at varying combinations and concentrations of growth regulators and reduced level of nitrogen concentrations, spots were produced at similar Rf values (0.10, 0.19, 0.27 and 0.35). This result revealed that these four compounds might be present in all the callus lines (Table 2).   Therefore, it is essential to conduct further studies for identification of fragrance compounds from the cell cultures of G. walla.

CONCLUSION
Based on the results of present study, ethyl acetate was selected as the best extracting chemical from callus and plant samples of agarwood while 15% methanol: chloroform was the best eluting solvent for silica gel TLC. TLC chromatographic finger prints revealed that there might be four similar compounds (Rf values of 0.60, 0.66, 0.78 and 0.87) which can be used as possible markers for G. walla, which were present in the ethyl acetate crude extracts of callus stem cuts, barks and agarwood. This preliminary study has proven that similar compounds as present in plant and agarwood of G. walla could be produced through plant cell culture techniques. Further investigations on two selected callus lines to identify the specific chemicals responsible for the peculiar fragrance of agarwood are essential to develop a protocol for the production of agarwood fragrance compounds through plant cell and tissue culture of G. walla.