Comparative Analysis of Latex Plants by GC-MS using Methanol Extraction

Abstract

Plants are able to produce a large number of diverse bioactive compounds. Solvent extraction is used for isolation of plant metabolites. The extract yield for plant metabolite extraction strongly depends on the nature of solvent. A review showed the methanol can yield more bioactive compounds. Drying of the sample material is also important for the extraction of plant material. The present study was carried out to analyze the phytocomponents of 5 different latex producing plants. The plants like Calotropis gigantea, Carica papaya, Nerium oleander, Ficus benghalensis and Plumeria alba leaves and latex. The GC-MS analysis of the metabolites revealed phytocomponents. Calotropis gigantea leaves showed 14 compounds and latex produced 5 compounds out of this 4,4,6A,6B,8A,11,11,14B-Octamethyl-1,4,4A,5,6,6A,6B,7,8,8A,9,10,11,12,12A,14,14A,14B-Octadeca-hydro-2 and 2R- Acetoxymethyl-1,3,3-trimethyl-4T-(3-Methyl-2-Buten-1-Yl)-1T-Cyclohexanol compound was present in both latex and leaf extraction. Beta. -carotene compound was present in both latex and leaf of Carica papaya. It was observed that Ficus benghalensis contained 2R-Acetoxymethyl-1,3,3-trimethyl-4T-(3-Methyl-2-Buten-1-Yl)-1T-Cyclohexanol was same in latex and leaf extraction.

Introduction

Medicinal plants are the backbone of traditional remedies.¹ The plants may contain many biological components with medicinal values and also be used for application purposes. It is the source for making new drugs.² Plants can produce a wide range of phytocomponents that can protect against free radicals that accumulate in fruits and vegetables. Phytochemicals are a collective term for both bioactive and non-nutritive plant substances. Recently, the use of phytochemicals has increased, especially in the areas of functional foods and pharmaceuticals.^3,4 There are about 10% of flowering plants that produce latex. Over 40 families are represented, including Euphorbiaceous, Apocynaceae, Caricaceae, Moraceous and Asclepiadaceae. Latex is a milky white fluid that is secreted by ducts of lactiferous tissue in leaves, stems, fruits and even in roots. some plants, like Euphorbia hirta, Euphorbia tirucalli, Jatropha gossypifolia, Plumeria rubra, Nerium oleander, Calotropis procera, Ficus benghalensis, Ficus religiosa and Carica papaya latex, were used to identify the phytochemicals present in it but not the methanol extraction study. The naturally synthesised chemical compounds are used to defend against predators like insects, fungi, and herbivorous mammals.⁵ Phytochemical screening is the method that has been used to detect antioxidant compounds in plants.⁶ Extraction is one method for extracting phytochemicals from the selective plant materials. The phytochemicals yield not only depend on the extraction technique but also depend on the solvent which is used for extraction.⁷ There are so many techniques involved in the extraction method, like maceration, infusion, percolation, microwave assisted extraction, the Soxhlet extraction technique, and also other extraction methods like accelerated solvent extraction and supercritical fluid extraction.⁸ (Figure 7 and 8) In this research, magnetic stirrer was used for extraction process. The primary goal of extraction is to extract as much of the specific compound as possible while obtaining the highest biological activity from the extract. Many solvents like methanol, ethanol, chloroform, acetone, and water, have been used for extraction depending upon the plant material. A review showed the highest extraction yield by methanol.

It has been proven to be more effective in the extraction of polyphenols with lower molecular weight. Methanol was found to be more effective than ethanol to extract a large number of phenolic compounds.² Likewise, (Figure 6) drying the plant material is also important because fresh plant materials may have active enzymes that produce the active constituent’s intermediates and metabolic reactions in the plants. Many researchers have done their research by drying plant material in an air-dry process in the shade in a dark room because overheating can cause the volatile substance and light-sensitive constituents to be lost.¹¹ Calotropis procera, Carica papaya and Ficus benghalensis latex were used for extraction by petroleum ether and methanol for antibacterial and antioxidant activity.¹²

Material and methods

Selected plants and collection

The leaves and latex of 5 different latex producing plants like Calotropis gigantea, Carica papaya, Nerium oleander, Ficus benghalensis, and Plumeria alba in Vellore district, Tamil Nadu, India.

Extract preparation

For latex, (Figure 1) incisions were made using a sharp, clean knife in the fresh green fleecy stem region of the Calotropis, Nerium, Ficus, Plumeria, and from papaya fruit. The latex was collected in a sterile vial, and immediately, the methanol was added to it to avoid a coagulation in a 1:1 ratio (Figure 2).¹²

Figure 1. Latex collection.

Figure 2. Latex with methanol.

For leaf extraction, the collected leaves were shade – dried (Figure 3) for 10 to 15 days to fully dry and then crushed into a powder using an electrical grinder. The dried leaf powder was then dissolved in 50 mL of methanol and placed in a magnetic stirrer set to maximum speed at 60 to 70℃ for 3 hours (Figure 4). The setup was kept without disturbance for 24 hours, and then the extract was filtered using normal filter paper. The filtrate was kept there for methanol evaporation in clear petri dishes, and the sample was sent there for GC-MS analysis.¹³

Figure 3. Shade dry.

Figure 4. A-Leaf sample with methanol, B-Latex sample with methanol.

GC-MS analysis

GC-MS analysis of the methanol extract of Calotropis gigantea, Carica papaya, Nerium oleander, Ficus benghalensis and Plumeria alba leaves and latex was performed using Clarus 680 GC employed a fused silica column, packed with Elite-5MS (5% biphenyl, 95% dimethylpolysiloxane, 30 m × 0.25 mm ID × 250 µm df). Helium was used as the carrier gas at a constant flow of 1ml/min using an injection volume of 1 µL. The injector temperature was set to 260℃ and the ion source temperature to 240℃, with a scan time 0.2 seconds and scan interval of 0.1 seconds. The spectrums of the components were compared with the database of spectra of known components stored in the GC-MS NIST (2008) library.

Result and discussion

The Identification of phytocomponents in plants has become more common in recent years because of their source of availability and their activity as bioactive compounds in various fields. Many studies have been undertaken to identify the compounds from the plants, but there are only a few latex producing plants that were used for analysis with methanol extraction. GC-MS analysis is a very good technique to identify the phytocomponents in the plant extract. In the present study, phytocomponents were taken from 5 different latex producing plants, namely Calotropis gigantea, Carica papaya, Nerium oleander Ficus benghalensis and Plumeria alba, of both leaf and latex.

Analysis of Calotropis gigantea leaf and latex:

The GC-MS technique revealed the result completely. The Calotropis gigantea plant contained 19 compounds, (Table 1) 14 of which were from leaf and (Table 2) 5 from the latex. This 2R-Acetoxymethyl-1,3,3-trimethyl-4T-(3-Methyl-2-Buten-1-YL)-1T-Cyclohexanol (Figure 5A) compound was observed at RT-30.325, with a molecular formula of C₁₇H₃₀O₃ and molecular weight of 282, and 4,4,6A,6B,8A,11,14B-Octamethyl-1, 4, 4A, 5, 6, 6A, 6B, 7, 8, 8A, 9, 10, 11, 12, 12A, 14, 14A, 14B Octadecahydro-2 (Figure 5B) was detected at RT 28.854, molecular formula is C₃₀H₄₈O, and its molecular weight 424, which were the same in both the leaf and latex methanol extractions and it is the major compound (Figure 5B) present in the latex at a concentration of 48.750% . However, (Figure 5C) the major compound in the leaf extract 3-Tetradecyn-1-ol was found at RT 20.851, molecular formula C₁₄H₂₆O and molecular weight 210 with the area 26.36 %. in leaf.

Table 1. Phytocomponents identified in the methanolic leaf extract of Calotropis gigantea by GC-MS.

Table 2. Phytocomponents identified in the methanolic latex extract of Calotropis gigantea by GC-MS.

Figure 5. Mass spectra of same and major compound in Calotropis leaf and latex. A)-2R-Acetoxymethyl-1,3,3-trimethyl-4T-(3-Methyl-2-Buten-1-YL)-1T-Cyclohexanol. B) 4,4,6A,6B,8A,11,14B-Octamethyl-1, 4, 4A, 5, 6, 6A, 6B, 7, 8, 8A, 9, 10, 11, 12, 12A, 14, 14A, 14B Octadecahydro-2. C) 3-Tetradecyn-1-ol.

The Calotropis gigantea plant latex and leaf were used for methanol extraction using the Soxhlet apparatus.^12,14 However, the compounds discovered in this study and those mentioned in the literature were not the same.¹⁵ has done the extraction of phytochemicals using Calotropis gigantea flowers not the methanol extraction; they identified the hexa-decanoic acid and squalene compounds which were the same in this present study in Calotropis gigantea leaves. These compounds showed anti-oxidant and anti-tumour biological activity.

Analysis of Carica papaya leaf and latex

In Carica papaya, GC-MS analysis showed total of about 20 compounds, of which (Table 3) 15 compounds were identified in leaf extract and (Table 4) 5 in latex methanol extraction. This Beta- carotene phytochemical had the same molecular formula C₄₀H₅₆ and molecular weight 536 in both leaves and latex at RT 29.314. The major compound in the leaf was hexa-decanal, with a RT of 21.156, a molecular formula C₁₆H₃₂O, a molecular weight of 240, and an area of 18.507 %. The main compound in latex was (1R,2R,8AS) -2,4,4,7A-Tetramethyl-1-(3-oxobutyl)-trans-hydrindan-2-carboxylic acid with a RT of 27.929, a molecular formula of C₁₈H₃₀O₃ and molecular weight of 294 and a peak area of 63.574%.

Table 3. Phytocomponents identified in the methanolic extract of Carica papaya leaf by GC-MS.

Table 4. Phytocomponents identified in the methanolic extract of Carica papaya latex by GC-MS.

Analysing the phytochemicals found in Carica papaya leaf and latex was one of the research. In the methanol extraction of papaya red lady variety leaf¹⁶ and in this investigation, it was demonstrated that no compound was same. However, the research revealed that n-hexadecanoic acid, which is present in methanol extraction of papaya leaf, is present. The same component was discovered in an aqueous papaya fruit extract that exhibited anti-microbial, anti-cancer, anti-haemolytic and anti-diabetic properties.¹⁷ Squalene was discovered in this study methanol leaf extraction; the same substance had previously been discovered by chloroform extracted latex.¹⁸

Analysis of Ficus benghalensis leaf and latex

GC-MS analysis of Ficus benghalensis revealed a total of 16 phytocompounds, (Table 5) 11 of which were found in the leaf and (Table 6) 5 in the latex after methanol extraction. 2R-Acetoxymethyl-1,3,3-trimethyl-4T-(3-methyl-2-buten-1-yl)-1T Cyclohexanol at RT 28.124, with a molecular formula of C₁₇H₃₀O₃ and a molecular weight of 282 was found in both the leaf and the latex of Ficus methanol extraction. The major compound in the leaf was N- hexadecanoic acid at RT 21.461, molecular formula C₁₆H₃₂O₂ and molecular weight 256, with an area of 20.907 % peak area. The latex main constituent was 6. Beta-bicyclo [4.3.0] nonane.5 beta-iodomethyl-1 beta-isopropenyl-4 with a RT 27.684, a molecular formula of C₁₅H₂₅I, and a molecular weight of 332 with a peak area of 53.278%.

Figure 6. Mass spectra of same and major compound in Carica papaya leaf and latex. (A) Beta- carotene, (B) Hexa-decanal, (C) (1R,2R,8AS) -2,4,4,7A-Tetramethyl-1-(3-oxobutyl)-trans-hydrindan-2-carboxylic acid.

Table 5. Phytocomponents identified in the methanolic extract of Ficus benghalensis leaf by GC-MS.

Table 6. Phytocomponents identified in the methanolic extract of Ficus benghalensis latex by GC-MS.

Figure 7. Mass spectra of same and major compound in Ficus leaf and latex. A) 2R-Acetoxymethyl-1,3,3-trimethyl-4T-(3-methyl-2-buten-1-yl)-1T Cyclohexanol, B) N- hexadecanoic acid, C) 6. Beta-bicyclo [4.3.0] nonane.5 beta-iodomethyl-1 beta-isopropenyl-4.

One of the study examined the phytocomponents in a methanol extract of Ficus benghalensis leaf. N-hexadecanoic acid was same in both studies in this as well. They state that this substance has anti-seborrheic, anti-inflammtory, cytoprotectant and anaesthetic properties.¹⁹ There are no studies was carried out using methanol extraction of Ficus benghalensis latex.

Analysis of Nerium oleander

GC-MS analysis of Nerium oleander revealed a total of 11 phytocompounds, (Table 7) 7 of which were found in the leaf and (Table 8) 4 compounds in the latex after methanol extraction. There is no similar compound found in Nerium methanol extraction leaf and latex. The major compound in the leaf was myo-inositol,4-c-methyl-at RT 20.185, molecular formula C₇H₁₄O₆, and molecular weight 194, with an area of 87.718% peak area. Lupeol was the major compound in the latex, with a RT of 28.474, a molecular formula of C₃₀H₅₀O, a molecular weight 426, and an area of 55.852% peak area.

Table 7. Phytocomponents identified in the methanolic extract of Nerium oleander leaf by GC-MS.

Table 8. Phytocomponents identified in the methanolic extract of Nerium oleander latex by GC-MS.

This research’s dl-alpha-tocopherol chemical compound, which is also found in the leaf extract, was compared to a study that examined the phytocomponent in Nerium oleander leaf part methanol extraction. They also emphasised the biological effects of this chemical, including its anti-oxidant, immunological, anti-cancer, anti-inflammtory properties.²⁰ There are no studies was carried out using methanol extraction of Nerium oleander latex.

Figure 8. Mass spectra of same and major compound in Nerium leaf and latex. A) myo-inositol,4-c-methyl, B) Lupeol.

Analysis of Plumeria alba

In Plumeria alba, GC-MS analysis showed total of 16 phytocompounds, of which (Table 9) 10 were found in the leaf and (Table 10) 6 compounds in the latex after methanol extraction. 2,4,4-Trimethyl-3-hydroxymethyl-5A-(3-Methyl-but-2-enyl)-cyclohexene at RT 28.069, with a molecular formula of C₁₅H₂₆O and a molecular weight of 222 was found in both the leaf and the latex of Ficus methanol extraction. The major compound in the leaf was tridecanoic acid at RT 19.160, molecular formula C₁₃H₂₆O₂ and molecular weight 214, with a peak area of 53.664 %. At RT 27.919, molecular formula C₁₅H₂₆O, and molecular weight 222, the major compound in the latex was 1-Methylene-2B-Hydroxymethyl-3,3-dimethyl-4B-(3-Methylbut-2-enyl)-cyclohexane with the area 52.524% peak area.

Table 9. Phytocomponents identified in the methanolic leaf extract of Plumeria alba leaf by GC-MS.

Table 10. Phytocomponents identified in the methanolic leaf extract of Plumeria alba latex by GC-MS.

Figure 9. Chromatogram of same and major compound in Plumeria leaf and latex. A) 2,4,4-Trimethyl-3-hydroxymethyl-5A-(3-Methyl-but-2-enyl)-cyclohexene, B) tridecanoic acid, C) 1-Methylene-2B-Hydroxymethyl-3,3-dimethyl-4B-(3-Methylbut-2-enyl)-cyclohexane.

One research did not analyse the methanol extraction, just the phytochemicals found in Plumeria alba flower. They named the substance Squalene.²¹ The Plumeria alba leaf in the current investigation contained the same chemical, as determined by methanol extraction.

Conclusion

The investigation in this study involves the leaves and latex of five different plants. As per the literature, this shows the presence of more identified phytocomponents and a few compounds that showed biological activity. The Calotropis gigantea plant latex and leaf were used for methanol extraction using the Soxhlet apparatus.^13,14 But the compounds in this study and those mentioned in the literature were not the same.² It has done the extraction of phytochemicals using Calotropis gigantea flower, not the methanol extraction; they identified the hexa-decanoic acid and squalene compounds which were found in this study in Calotropis gigantea leaf, and Carica papaya leaf, and Plumeria leaf. These compounds showed the antioxidant and anti-tumour biological activities. Beta carotene, which is present in the Carica papaya leaf as well as in the latex, the was shown in the review to play a dynamic role in delaying aging, reducing inflammation, and preventing certain cancers.¹⁹ Literature showed that identified compound phytol in ethanolic leaf extraction of Calotropis gigantea that showed anti-inflammatory and anti-cholesteric activity, the same was identified in this study in Carica papaya leaf. The comparison of latex and leaf methanolic extraction reveals a large number of compounds, with some compounds present in both leaf and latex.