Steroid and Fatty Acid Contents from the Leaves of Carica Papaya

Aim: To extract and identify the non-polar entities from the leaves of Carica papaya, a plant used for medicinal purpose as folk medicine. Materials and methods: Petroleum ether extract of the Carica papaya leaves was used for this study. Saponification process and methylation process was performed to separate fatty acids and unsaponifiable matters. Phytochemical constituents were separated using chemical process and separated fractions were analyzed by thin layer chromatography (TLC) and gas chromatography coupled with mass spectroscopy (GC-MS). Results: The chemical composition of the steroids, triterpenoids and fatty acid methyl esters (FAMEs) in leaves of Carica papaya, which were analyzed by gas chromatography coupled with mass spectroscopy (GC-MS). A total of 15 fatty acid components were identified in saponifiable matter, from unsaponifiable portion 2 steroids (campesterol, βor γ-sitosterol), 1 triterpene (squalene), and 1 diterpene (phytol) were identified. Conclusions: The results indicate that the extract is rich in non-polar compounds. In this study, GC-MS method is at the central focus for identification of these phytoconstituents. The current method can be used for direct analysis of non-polar entities of plant material.


AIM
For identification of the non-polar component, first, it is necessary to separate lipids from the plant material. Many classical methods and references are readily available for the extraction and identification of steroids and terpenoids from other medicinal plants [19][20][21][22][23] ; yet it is necessary to determine a specific method to isolate these molecules or fractions which ought to be standardized.

Plant materials
Fresh leaves of Carica papaya were collected from Rajkot (Gujarat), India in winter, 2019 and were authenticated by the Department of Botany, Shri M. & N. Virani Science College (Autonomous), Rajkot, Gujarat, India. The leaves were prewashed with distilled water to remove dust particles and shed dried for three days at room temperature and grinded to obtain powder (144 gm) for extraction purposes.

Preparation of extract
The leaves powder (20 gm) was extracted with petroleum ether (60°-80°C) by continuous Soxhlet's extraction method for 12 hours (52 cycles). The extract was concentrated and dried under reduced pressure, which yielded a brownish mass (2.3 gm). This crude extract was utilized for further experiment.

Saponification of petroleum ether extract
The crude extract of petroleum ether (2.3 gm) was taken in RBF and 7.5% methanolic KOH (50 ml) was added to it. The resultant mixture was refluxed for 5-6 hours, upon cooling at room temperature; distilled water (20 ml) was added. The mixture was then extracted with diethyl ether, until diethyl ether remains transparent after partition process (liquid-liquid extraction).The combined diethyl ether extract was dried over anhydrous sodium sulphate (Na 2 SO 4 ) and evaporated to obtained unsaponifiable fraction (108 mg) (fraction A).
The aqueous portion left after ether extraction was labeled as saponifiable portion, which was acidified with 5N H 2 SO 4 and the aqueous layer was further extracted 3-4 times with diethyl ether. Ether layer was partitioned with distilled water and extractive was dried over anhydrous Na 2 SO 4 , evaporated to obtain fatty acid portion (144 mg) (fraction B).

Investigation of unsaponifiable fraction (fraction A)
The unsaponifiable fraction (fraction A) was tested with Liebermann-Burchard reagent for the confirmation of steroid and triterpenoids present by thin layer chromatography (TLC F 254 ) with solvent system chloroform: methanol (9.5:0.5) which gave a strong positive test, and then it was analyzed by GC-MS.

Methylation of fatty acid
An accurately weighed portion of (fraction B) fatty acid (140 mg) was taken into RBF and refluxed in water bath for 5 hours with solution of methanol: benzene: conc. H 2 SO 4 (43:5:2). Cooled the same and diluted with water (40 ml) and extracted with n-hexane until the green color stop extracted. The combined extract was dried over anhydrous sodium sulphate and studied further for GC-MS analysis.

Instruments
GC-MS analysis was carried out on a Shimadzu GC-MS (model no. TQ8040) sampler and gas chromatograph interfaced with mass-spectrometer (GC-MS) instrument employing the following conditions.

GC-MS protocol for unsaponifiable fraction (fraction A)
SH-Rxi-5Sil MS capillary column (30 m×0.25 mm ID×0.25 μM df), operating in an electron impact mode at 70 eV; helium (99.999%) was used as carrier gas at a constant flow of 1 ml/min and an injection volume of 0.5 μl was employed (split ratio of 5:1); injector temperature 250°C; ion-source temperature 290°C. The oven temperature was programmed from 100°C (isothermal for 2 min), with an increase of 15°C/min, to 180°C, then 5°C/min to 300°C, ending with an isothermal at 300°C. Mass spectra were taken at 70 eV; a scan interval of 0.5 seconds and fragments from 45 to 450 Da.

GC-MS protocol saponifiable fraction (fraction B)
SH-Rxi-5Sil MS capillary column (30 m×0.25 mm ID×0.25 μM df), operating in an electron impact mode at 70 eV; helium (99.999%) was used as carrier gas at a constant flow of 1 ml/min and an injection volume of 0.5 μl was employed (split ratio of 5:1) injector temperature 100°C; ion-source temperature 250°C. The oven temperature was programmed from 50°C (isothermal for 2 min), with an increase of 10°C/ min, to 100°C, then 15°C/min to 280°C, ending with an isothermal at 280°C. Mass spectra were taken at 70 eV; a scan interval of 0.5 seconds and fragments from 45 to 450 Da.

RESULTS
Interpretation of GC-MS was conducted using the similarity search from the database of National Institute Standard and Technology (NIST). The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST 17 Library having >260,000 patterns.

Analysis of unsaponifiable fraction (fraction A)
The R f value of the track at 254 nm, 366 nm and post chromatic derivatization with Lieberman-Burchard spray rea-  gent are presented in Table 1. It revealed that from sample, 10 components were observed at 254 nm and 5 components were observed on 366 nm at different R f values. After sprayed with Lieberman-Burchard reagent it was confirmed as a steroid or triterpenoids compounds (Fig. 1). An unsaponifiable fraction was injected into GC-MS and two steroids, one triterpene, and one diterpene compound were identified and confirmed the result by GC-MS (Fig.  2). The comparison of obtained mass spectrums with the NIST library database gave us a confirmatory structure of compounds.

GC-MS fragment
The

Analysis of saponifiable fraction (fraction B)
The major fatty acid components were identified by noting their retention time and comparing it with the retention time of an authentic sample of methyl ester of fatty acid. GC analysis of the ester residue showed several peaks (Fig.  7). Table 2 shows that 20 compounds were identified in Carica papaya leaves. Major FAMEs fraction was identified as 2-methylpentane (11.99%), 3-methylpentane (11.72%), acetyl valeryl (37.49%), methyl-cyclopentane (19.55%), and other identified compounds.

DISCUSSION
Qualitative test of extract gave positive test with Lieberman-Burchard reagent, which suggests the presence of steroid or triterpenoids. TLC results revealed that unsaponifiable load in Carica papaya was higher. The unsaponifiable matter was separated through extraction with non-polar solvents like diethyl ether. From the unsaponifiable fraction, one triterpene, one diterpene and two steroid compounds were identified in saponifiable fraction; the mixed fatty acids were liberated by addition of 5N sulphuric acid. It was difficult to separate higher fatty acids through simple chemical methods; hence, they were converted into their methyl esters which were amenable to separation through various chromatographic techniques like adsorption TLC, reversed phase TLC and GC-MS.
In the present study, GC-MS technique was employed to study the unsaponifiable and saponifiable matter in the test plant. The comparison of the mass spectrums with the database gave more than 95% match as well as confirmatory compound structure match.

CONCLUSIONS
In the present study, we have extracted and identified fifteen fatty acid components in saponifiable fraction and two steroids (campesterol, β-or γ-sitosterol), one triterpe-ne (squalene), one diterpene (phytol) from the unsaponifiable fraction of Carica papaya leaves; it is the first time reporting in this plant. These results indicate that the extract is rich in non-polar compounds. In this study, GC-MS method is at central focus for identification of these phytoconstituents. This method is a direct and fast analytical approach for identification of non-polar entities of plant material, and also this method can be applied in the analysis of any other low concentration minor component fatty acids in complex sample matrices.