Current World Environment

Introduction

Increasing energy demands due to rapid industrialization, expansion of agriculture and enormous increase in population have not only resulted in the extensive depletion of non-renewable petroleum resources but also increased global warming and climate change. This environmental deterioration and loss of fossil fuels has promoted the research in search of alternative renewable energy. Hence, it is essential to look for new sources of organic compounds which can act as substitute of petroleum-based substances.^1,2 These sources are plants which are called Petrocrops as they produce petroleum substances as supplementary to petrol. These plants produce hydrocarbon compounds or ethanol. Calotropis procera, Pedilanthus tithymaloides, Copifera langsdorfii, Euphorbia lathyris and Jaropha curcas are examples of petro-crops. It has been reported that gasoline and identical compounds are obtained from plant hydrocarbons with the help of catalyst like  Zeolite (ZSM-5 type).^3,4In Grindelia squarrosa (Pursh) whole plant methylene chloride extract has been reported to be highly converted into liquid fuels⁴. Such closely developed methods change the scope of fuels and biochemicals. 

Most of the phytochemical crop studies by Calvin,⁵  are based on latex- bearing species, particularly Euphorbia species that can also be grown in arid and semi-arid regions of the world. A popularly known ornamental plant Pedilanthus tithymaloides (L.) Poit. (Euphorbiaceae), new scientific name Euphorbia tithymaloides (L.) Poit. is a succulent shrub commonly found in America and Asia. It is 0.4m to 3m tall and 40-60 cm wide. Some common names for Pedilanthus tithymaloides include Red  Bird Flower, Devil's Backbone,  Bock thorn, Christmas Candle, Fiddle Flower, Slipper  Plant, Violin Flower, and so on. It thrives in various soil types, especially those with abundant micronutrients like Boron, Copper, Iron, Manganese, Molybdenum and Zinc. while it struggles to grow in highly saline soil. The plant can flourish when supplemented with fertilizers. Stem is olive-green with ovate leaves and beak-shaped flowers. laticifers are present in the whole plant. It is known as petro-crop and has medicinal importance also. Investigations have been carried out in the field of developing agro-technology for enhancing biomass and bio crude productivity of Pedilanthus tithymaloides.^6,7,8,9,10 There was a need to develop proper extraction technique and to identify the hydrocarbon compounds present in the plant. Research was carried out on extraction procedure of hydrocarbons in the University of Arizona. Analysis of latex has revealed the presence of a large number of secondary metabolites in Calotropis  procera ^{11,12,13,14,15,16} Euphorbia sikkinensis Boiss ¹⁷Croton bonplandianum Baill¹⁸ , Euphorbia hirta ^19,20,21,22Euphorbia lathyris ²³Major classes of hydrocarbon like substances present in bio-crude include terpenoid, isoprenoid, polymers and long chain aliphatics. Hydrocarbons from Pedilanthus macrocarpus have been analyzed by H’-NMR indicating the alkane range C27H56 to C35H72²⁴. Gas-liquid chromatography of E. antisyphilitica revealed the presence of C29H60, C31H62 and C33 H68 compounds²⁴. A new tetracyclic triterpene (3-epi-cyclolaudenal-22-en-3 alpha-ol) cyclolaudenol has been reported in E. cauducifolia.²⁵Alcoholic extract of roots of E. ferganensis B. Fedtsh yielded on chromatography an aromatic ethyl ether (C29 H1008, m.p. 158-160 degree Celsius) and a coumarin, scopoline .²⁶

 Phytochemical analysis was carried out on P. tithymaloides var. green, var. cuculatus and var. variegatus to find out the occurrence of various metabolites that might have potential pharmaceutical applications as well as use in biomass derived hydrocarbon like material of high-grade transportation fuel.

Materials and Methods 

Plants of P. tithymaloides var. green, var. cuculatus and var. variegatus were harvested in the month of August and identified in the herbarium of Rajasthan University. Aboveground portions were dried and powdered.  Thimbles of this powder were used for soxhlet extraction using low-boiling nonpolar hexane as solvent. The hexane extract of P. tithymaloides var. green obtained was then fractionated using the column chromatographic technique. Some of the fractions thus obtained were subjected to gas-liquid chromatography at the University of Rajasthan.   

Column chromatography 

Silica gel H Glaxo grade was used for the packing of the column measuring 1 cm x 60 cm. The bio crude obtained by soxhlet extraction was filtered twice or thrice on Whatman filters paper No. 1. The filtrates obtained were mixed with silica gel and loaded on a packed silica gel column. This was covered by a thin cotton layer. Different solvents were passed through the column serially according to their increasing polarity. The solvents used in the present study were hexane (having polarity 0.0), benzene (having polarity 2.7), ethyl acetate (having polarity 4.4), acetone (having polarity 5.1), methanol (having polarity 6.6) and chloroform (having polarity 9.4) in a sequence in the combination of pure solvent, 3:1, 1:1 and 1:3. Twenty-one fractions were collected from the extract based on colour.

Gas Liquid Chromatography 

The fractions obtained from column chromatography were studied using the GLC technique. The chromatography used was Hewlett-Packard 5890A gas-liquid chromatography.  0.5µl fraction was injected on the 10-meter long HPL (Methyl Silicon gum column) of 0.53 mm diameter with a film thickness of 2.65 µm. The chart speed was 0.50-1.0 cm. per minute. Nitrogen was used as carrier at a flow rate of 40 ml/minute.  The flow of hydrogen was 35 ml/minute and of air was 360 ml/minute. The column was initially set at 50?C and it gradually increased to a final temperature of 300?C at a rate of 15?C per minute.

Qualitative phyto-chemical estimation of  biocrude  and plant dried samples–

To estimate the occurence of different hydrocarbons in the biocrude and plant-dried samples qualitative phyto-chemical assay was carried out using established methods proposed by Gibbs²⁷ and Harbone.²⁸

Results and Discussion

In the present investigation ground plant parts including leaves and stems of all  three varieties of Pedilanthus tithymaloides collected in urban areas were processed to obtain the profile of the phytochemicals present.  The assay resulted in the detection of Flavonoids, Triterpenoids, Sterols, Alkaloids, Anthraquinones and Saponins. However, Tannins, Iridoids, Juglone, Leucoanthocyanins and Catechol were not detected (Table 1).

Column chromatography fractions of the bio crude when analyzed through GLC revealed peaks at different retention times for a large number of compounds that need further characterization. Fraction obtained in hexane: benzene (1:1) showed four major peaks at retention time 4.90, 5.10, 6.76 and 8.86 minutes at chart speed 1.0 cm/per minute. Fraction obtained in benzene and fraction obtained in benzene: ethyl acetate in the ratio of 1:1 and 1:3 showed various peaks. Major peaks obtained were at 2.98, 4.36, 5.71, 6.79, 8.76 and 9.11 minutes at chart speed.1.0 cm/min. in the fraction obtained in pure ethyl acetate. Gas-liquid chromatograms (1 -5). 

Table 1: Response of Pedilanthus  tithymaoloides var. green, var. cuculatus and var. variegatus to different phytochemical tests.

Figure 1: Gas-liquid chromatograms (1 -5).   Click here to view Figure

Other workers also found similar results as hexane, ethyl acetate and ethanol extracts from leaves of Pedilanthus tithymaloides were tested phytochemically resulting in the detection of triterpenes, steroids, saponins, tannins and coumarins which were effective for antimicrobial activities and other biological properties.^29,30,31. Sandjo and coworkers³² reported that nine coumarin derivatives derived from Pedilanthus were able to stop the germination of Magnaporthe orizae a phytopathogenic sporulating fungus. Antimicrobial activities of diterpenoids extracted from Pedilanthus tithymaloides were reported to be effective against Mycobacteryum tuberculosis .³³ The major components extracted from Euphorbia latex were tetracyclic triterpenoids mainly cycloartenol, 24-methylene cycloartenol and lanosterol. ³⁴ It was reported that the latex of E. lathyris  converts acetate and mevalonate to many tetracyclic triterpenes, mainly triterpenols and their fatty acid esters which are structurally different.^35,36 Detailed investigations carried out to find out the source of triterpene biosynthesis have revealed that an osmotically sensitive organelle of the plant cell and possibly calmodulin are involved in the triterpene biosynthesis. ^34,37,38,39

The experiments show that triterpenol and triterpene esters are synthesized at separate sites in a plant. Triterpene ester with triterpenols of the latex are synthesized in the wall lining cytoplasm of the laticifers in Hoya diversifolia Blume. and other Hoya species, (Asclepiadaceae), ^40,41,42 

An assortment of solvents were utilized to extract pertinent phytochemicals, such as coumarins, tannins, steroids, triterpenes, and saponins from hexane and ethyl acetate extracts⁴³. Kaempferol 3-O-o-D-glucopyranoside-600-(3-hydroxy-3-methylglutarate), quercitrin, isoquercitrin, and scopoletin were the antioxidants found in a bioassay. A total of 76 point 0 mg of gallic acid equivalents/g extract ⁴⁴ was determined to be the phenolic and flavonoid contents. Pedilanthus tithymaloides was found to contain a set of four coumarin derivatives.⁴⁵ Steroids, cardenolides, anthraquinones^, alkaloids, unsaturated steroids,  phenolics, leucoanthocyanin etc. were reported from Pedilanthus tithymaloides.⁴⁶ Plant chemicals such as flavonoids, terpenoids, phenols, tannins, saponins, glycosides, sterols, amino acids, and reducing sugars were found in the water filtrate of Pedilanthus leaves and stems. These  secondary metabolites are useful in the creation of substitute bio-control agents that target mosquito vectors and agricultural insect pests.⁴⁷  

Twenty-one latex-bearing plants in the Indian state of Maharashtra were the subject of analysis. It was found that the secondary metabolites of these plants, which included  Pedilanthus  tithymaloides,  were primarily alkaloids, flavonoids, terpenoids, cynogenic glycosides, phenolics, tannins, and saponins. These findings are consistent with the current study. The main finding was that all latex-bearing plants possessed pharmaceutically important secondary metabolites.⁴⁸ Hence, the experimental plant has  pharmacognostic and energy potential. 

Conclusion

The findings derived from phytochemical analysis of the three varieties of P. tithymaloides revealed that the plants possess flavonoids, anthraquinones, sterols, alkaloids, triterpenoids and saponins. In order to determine the presence of gutta, rubber, waxes, and other materials, a gas-liquid chromatogram of hydrocarbon compounds extracted in hexane, benzene, and ethyl acetate revealed a number of peaks that could be further identified by ¹H NMR analysis,  ¹³ C-NMR analysis, IR spectroscopy, Proton Magnetic Resonance  (PMR)  and Gel Permeation Chromatography (GPC). The plant species is a very powerful energy source because of the plant's readily extracted compounds, such as triterpenoids, which can be cracked to produce high-octane gasoline. It can be used as a feedstock for fuel, chemicals, and petroleum derivatives. All three varieties are promising for development as petrocrops as they provide high biomass and high hydrocarbon yield. Evidence from the literature also justifies the pharmacognostic potential of the plant for treating various ailments.

Acknowledgements

The author would like to acknowledge the support of Rajasthan University Jaipur and his work place.

Funding Sources

The author received no financial support for the research, authorship and/or publication of this paper.

Conflict of Interest

The author doesn’t have any conflict of interest in the research.

Author’s Contribution

The research and paper writing was performed by the author itself.

Data Availability Statement

The manuscript incorporate all datasets produced or examined throughout this research study.

Ethics Approval Statement-

Experiment on no human or animal was performed during the study.

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