Chemical investigation of epicuticular wax obtained from Euphorbia milii leaves

Epicuticular wax analysis was performed on the leaves of chloroform extract obtained from the plant Euphorbia milii (‘Christ’s plant’). Aim of the study is to identify the chemical constituents and to discover how they were distributed within the cuticle. Column chromatographic separations based on polarity and GC–MS analysis led to the identification of the pentacyclic triterpenoids, its acetates and hydrocarbons that are found to be present in the epicuticular wax. The study revealed that the plant wax contains Lupenone, Glutinol, Lupeol acetate, Glutinyl acetate, Friedelan-3-ol,D:A-Friedooleanan-28-acetate 3beta hydroxyl in fraction1. The second fraction was found to contain Friedooleanan-3-ol, Friedooleanan-3-aceteate along with hydrocarbons of carbon chain length from C23–C33. Hydrocarbons in the form of alkanes and alkenes were identified as the major constituents in the third fraction of the leaf extracts and found to vary from carbon chain C18 to C34. Cuticular wax was found to be dominated by triterpenoids in the first two fractions. The last fraction contains hydrocarbons as the major constituent. The functional group analysis through FTIR-HATR study reveals the presence of characteristic peaks of waxes. The finding suggests that the biomass of the plant Euphorbia milii can be an important source for hydrocarbon.


Introduction
The aerial parts of most plants are covered by the thin layer commonly called cuticle. This consist of long-chain hydrocarbon compounds including alkanes, primary alcohols, aldehydes, secondary alcohols, ketones, esters and the other varying proportions of cyclic compounds like pentacyclic triterpenoids and hydroxycinnamic acid derivatives [1,2].Plant cuticle wax plays an important role in preventing the non-stomatal parts from loss of water through epidermis. It acts as a source for transpiration barrier and also protects the plants from the ultra violet rays like UV-A and UV-B in which the region of spectrum ranges from 280-320 nm. When plants are exposed to the dusty environment, it becomes more responsive to abiotic stresses such as temperature, physical damage, osmotic stress, pollution and altitude. The Cuticular wax layers respond to these stresses by providing a protective barrier on the leaf [3,4]. From the literature it is evident that the presence of n-alkanes in the leaf wax contributes to the hydrophobic property which helps the leaves to prevent the Cuticular water loss.
The important function of epicuticular wax layer include waterproofing from plants surface, control of Cuticular transpiration, protection against insect pest, foliar pathogens and reflectance or scattering of excessive incident radiation [5][6][7]. It is also reported that plants tends to produce a large quantity wax in the drought conditions to compensate the loss of water [8]. Epicuticular wax crystal on floral stems of certain plants prevent ants from removing nectar. Plants grown in-vitro was found to have reduced level of surface wax compared with the greenhouse counter parts due to the high relative humidity in the culture vessels [9]. The reduced wax layer was thought to be one of the reasons for desiccation and limited survival of these plants after transferring to greenhouse condition. The lower light intensity and high relative humidity have been shown to be correlated with lower production of epicuticular wax on plants obtained under these conditions [10]. In the recent studies, it is observed that the plants containing higher percentage of wax components are looked upon for its application to the hydrocarbon fuel production.
The present study addresses the evaluation of epicuticular wax content of the plant Euphorbia milii, commonly called as 'Crown of Thorn' or 'Christ plants' . The plant belongs to the family Euphorbiaceae, Genus Euphorbia, and Species Euphorbia milii. The plant has been used for the ornamental purpose and there is, however, a reason for renewed interest in this plants at a time when the emphasis is being placed on water conservation and ease to maintenance.
Euphorbia milii which is classified as succulent is native to Madagascar and also found in India and Kerala. This is a plant with thick fleshy leaves and stem are adapted to water storage. The leaves tend to be ovate and wider near the tip up to 1 and a half inches in length. Different species of euphorbia have been widely used in folk medicine for treatment of diarrhoea, inflammation, and swellings and is known as wart remover. Euphorbia milii crude latex showed to be a potent plant for molluscicidal and found to be toxic to animals [11]. Phytochemical studies on methanol extract of Euphorbia milii leaves revealed the presence of beta-sitosterol, cycloartenol, beta-amyrin acetate, lupeol, euphol, triterpenes, and flavonoids [12,13].E,hirta reported to contain hydrocarbons like paptacosane,nnonacosane in addition to triterpenoids [13]. Euphorbia is also reported for its anti-arthritis, anti-cancer [14][15][16], anticonvulsant,anti-diabetic, anti-eczema, anti-inflammatory, anti-microbial, anti-oxidant, anti-spasmodic, anti-tumour, anti-tussive, hormonal and myelopoiesis properties [17,18]. Preliminary anti-nociceptive studies of methanol extract of Euphorbia milli reveals the use of the plant as a pain reliever [19].
Hydrocarbon distribution in epicuticular waxes of five euphorbia species have been reported earlier [20] which demonstrated the presence of branched hydrocarbons when extracted with hexane. However the complete report on the wax content of Euphorbia milii leaves is not discussed as the study of the hexane extract obtained for five species which belong to the Euphorbia genera.
Despite of the report on the leaves of Euphorbia milli, the wax content of the leaves is not explored in detail. As the plant is characteristic of having thick leaf surface which is characteristic of having cuticles enriched with secondary metabolites, the objective of the study is to evaluate the epicuticular wax composition of the leaves and its responses to the environmental stress through FTIR-HATR studies. To the best of our knowledge, this is the first report on epicuticular wax content of Euphorbia milli leaves and its property as an abiotic stress tolerant plant.

Preparation of wax extract
About 2 kg of Euphorbia milii leaves were collected from VIT University nursery during the month of January. The fresh plants of Euphorbia milii have been authenticated by Prof. P. Jayaraman, Director, Institute of Herbal Science Plant Anatomy Research Centre, Chennai. For the extraction of epicuticular waxes, the leaves collected were washed with tap water for several times, left for shade drying. Then the leaves were run for extraction of epicuticular wax by immersion method using chloroform as the solvent. About 30 ml of chloroform was used for extraction initially in a petri dish followed by immersion of 5 to 10 leaves for about 30 s and the same procedure was repeated for 2 kg of leaves. The extract was then dried with nitrogen steam at 40 °C. The total amount of chloroform used was about 1000 ml for extraction of 2 kg of leaves. Then the extract was concentrated under rota evaporator to recover the excess solvent. The concentrated wax extract was then kept for complete evaporation at room temperature for few days. The amount of epicuticular wax extracted was found to be 55.9 g.The crude wax sample was then stored in a refrigerator until further use.

FTIR-HATR studies
A non-destructive Horizontal Attenuated Total Reflectance (HATR) -FTIR technique was used to study the surface response of the wax to the effect of temperature on the plant wax. The fresh leaf surface was subjected to HATR-Infrared spectral studies which were obtained with Nicolet Magna 550 FTIR Series II Spectrometer. The spectrum was recorded in the wavenumber range 4000-400 cm. using ZnSe crystal. To study the responses of the plant to the abiotic stress with respect to the temperature the experiment was designed to screen the wax content for the period of three months; January to March. This was compared with the same plant grown in the nursery which was treated as a control sample.

Phytochemical analysis
The crude extract of the wax was subjected to routine phytochemical tests to identify the presence of various constituents by following the methods described by J.B Harbone [21].

GC-MS studies
The GC-MS analysis was carried out on Perkin Elmer clarus 680 GC-MS instrument employing the condition viz; column elite-5MS (30.0 m, 0.25 mm ID, 250 μm, operating in electron impact mode; the carrier gas used was helium at a constant flow and split ratio of 10:1;the injector temperature was kept at 250 °C and the flow rate was constant at 1 ml/min; oven temperature was initially 60 °C for 2 min, ramp 10 °C/min to 300 °C, hold for 6 min. The total run time was set as 45.00 min. The molecular mass identification was done using the database of National Institute Standard and Technology (NIST).

Column chromatography separation
About 10 g of the crude sample of Euphorbia milii wax extract was subjected to column chromatography packed with silica gel (60 mesh) using hexane. The solvent systems used to elute the column were hexane (100%, 100 ml), Hexane: Ethyl acetate (9:1200 ml), Hexane: Ethyl acetate (7:3200 ml) and finally, the column were washed with methanol. About 50 fractions were collected in a test tube and grouped based on their Rf values and were named as F1, F2, and F3 fractions.

Micro-column chromatography
The F3 fraction which gave multiple spots in TLC was again separated by micro-column chromatography and gave rise to 14 fractions and they were again subjected to TLC. Based on the Rf values the compounds were further separated and named as F3a. The fractions were kept for drying at room temperature until for further characterization.

Phytochemical analysis
The plant epicuticular wax obtained by immersion method with chloroform as the extracting solvent was subjected to systematic phytochemical analysis. The preliminary phytochemical study reveals the presence of carbohydrates, flavonoids, tannins and triterpenoids in the epicuticular wax. (Table1).On comparing the results of the phytoconstituents with the earlier study on the same plant wax [20] it is found to contain additional plant components such as carbohydrates, flavonoids, tannins and triterpenoids. This may be due to extracting medium chloroform that is used in the present study, which is slightly polar than hexane.

FTIR-HATR studies
The FTIR-HATR scanning of leaves of Euphorbia milii showed absorption band at 3390 cm −1 due to the presence of O-H groups of alcohols or phenols. The band at 2916-2953 cm −1 revealed the present of the alkane group. This clearly indicates the presence of alkane groups which can help the plant to protect the leaves from water loss. The bands at 1730 cm −1 shows the presence of C = O group which is due to alpha-beta unsaturated ester. This demonstrates the presence of the wax esters in Euphorbia milii.
The presence of all these compounds has been confirmed by the following study.

GC-MS studies
As it is evident in the previous studies that the plant E.milli. contains rich amount of wax content, the present work aimed for the comprehensive analysis of wax content of the leaf obtained from E.milli. The GC-MS studies of the crude extract revealed the presence of homologous series of alkanes with odd numbers and mass spectrum further revealed the presence of pentacyclic triterpenoids and its esters.

Discussions
The nondestructive FTIR-HATR spectral studies of the Euphorbia milii leaves shows characteristic spectroscopic markers of waxes at 3200-3400 cm −1 ,intense peak at 2845-2925 cm and 1720-1730 cm −1 confirms the peaks due to O-H stretch, C-H, and C = O stretch respectively. Further on comparing the FTIR-HATR characteristic spectral intensity with the plant that is grown in nursery which is treated as the control, it was found that the plant grown outside the campus contains the same functional group and its increased intensity reveals the ability of the plant leaves to respond to the environmental stress. From Table 2, FTIR-HATR data of the leaves showed the characteristic band at 3200-3400 cm −1 confirming the alkanes (2845-2925 cm −1 ) present in the plant leaf samples helps to protect the leaves from water loss even during the dry season. The peaks were observed during the entire period of the study confirming the ability of the plant to with stand the temperature even during the summer. The band at 1730 cm −1 shows the presence of C = O group which is alpha-beta unsaturated ester. This is present in all the months revealing that Euphorbia milii is rich in epicuticular wax.All these observations make us to conclude that the plant could support the fact that it is could be recommended as abiotic stress tolerant plant. The epicuticular wax content of the Euphorbia milii leaves was further evaluated by the GC-MS studies which are discussed below.
The first fraction F1 obtained from the Hexane: Ethyl acetate (9:1) as the mobile phase showed six peaks in GC-MS (Fig. 1). This fraction revealed the presence of The second fraction F2 was obtained from Hexane: Ethyl acetate (7:3) as amoblie phase. GC-MS (Fig. 2) of the F2 fraction showed nine peaks which are characterized as follows; (1)Tricosane:C 23 H 48 ,(2) Octacosan:C 28 H 58 ,(3)  (Fig. 3). This fraction contained one unidentified compound. The hydrocarbons identified in all the three major fractions are given in Tables 3 and 4.
The study demonstrates that the composition of epicuticular wax obtained from the leaves of Euphorbia milii is found to be wax esters, pentacyclic triterpenoids and hydrocarbons. Fraction 1 is dominated with pentacyclic compounds which renders the property for the protection of the plant from the microorganism and harmful insects as reported in the literature. Thus, the presence of the phytoconstituents identified in the leaf wax confirms their important role in protecting the leaf from air pollution and the other environmental factors. The phytochemicals reported here confirms the availability of the chemical constituents reported in the literature which belong to the Euphorbiaceae family [13]. The study also justifies the uses of this plant not only as an ornamental plant but also can be grown in draught where to protect the water bed on the ground. Fraction 2 and 3 contains hydrocarbons as the major constituents which are expected to contribute to the water balancing property of the leaves of E. milii plant. In particular F3 is found to be dominated with the constituents of hydrocarbons.

Conclusion
To conclude the present study reveals that the wax content of Euphorbia milii leaves composed of hydrocarbons, wax esters, alcohols and ketones. This chemical composition of epicuticular wax proves that the wax coating on the leaves can protect the plant by preserving the water balance by reducing the evaporation from the leaf surface. The presence of hydrocarbons along with other waxy components is expected to serve as barrier to the passage of water in and out of the cell. This import and dominating constituent of the plant wax is found to be the alkanes and alkenes and this constituent is expected to prevent water inundation or dehydration as reported in the literature. Samples will be taken at large scale in E. milli and the study will be carried out to establish the reproducibility of the results and replicate study with respect to plasticity and adaptability of the species with reference to cuticular wax.
Funding The authors are thankful to VIT management, Vellore for providing necessary facilty to carry out this work.

Conflict of interest The authors don't have any conflict of interested.
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