Isolation of the isopropanol extract components of the leaves of Ailanthus glandulosa by a new method

The leaves of Ailanthus glandulosa were extracted with the Soxhlet apparatus using isopropanol. A new method was used to separate and isolate eleven chemical compounds present in the leaves of bird's tongue. The separation was carried out by column chromatography using displacement solvents (petroleum ether, chloroform, dichloromethane, methanol) and four eluates were obtained. The four eluates were treated with a number of solvents, yielding thirty-four compounds. The chemical content of the mordants was determined using GC/MS technology. The samples tested contained six ester compounds, three aldehyde compounds, three ketone compounds, two alcoholic compounds, eight carboxylic acid compounds, five silicone compounds, five aromatic compounds and one phosphate compound. Eleven compounds were isolated, the most important of which are: 2-naphthoxyacetic acid, 2,6-bis (1,1-dimethylethyl)-4-ethylphenol, 2,5-tert-butylnitrobenzene, 5-hexyl-2-furaldehyde, 16-nitrobicyclo [10.4.0] hexadecan-1-ol-13-one, cyclooctasiloxan hexadecamethyl.


Introduction
Ailanthus glandulosa, scientific name: Ailanthus altissima. It belongs to the samrobiate family and is a widespread deciduous forest tree found in temperate climates on both sides of roads, in wastelands and in abandoned areas. Its leaves are compound, green in colour and fall off in winter [1]. It belongs to the genus Ailanthus in the family Simarobica. It is called by many names depending on the country of origin, such as Tree of Heaven, Chinese Sumac, Copal Tree and Brooklyn Palm, Hell Tree. In the Middle East, this tree is known as Chinese Azdoracht or Chinese Azurekh. It is native to north-eastern and central China and Taiwan [2]. In many areas it is called the tree of hell because it grows very fast and can clone itself.
Researchers have demonstrated the effectiveness of the methanol extract from the leaves of Ailanthus glandulosa against free radicals (antioxidant) [7]. Some studies have also shown that the aqueous extract from the leaves of the Ailanthus glandulosa tree has antioxidant properties [8]. One study showed that the bark extract with petroleum ether had a strong repellent and killing effect against four major insects in shops and warehouses [9]. Other researchers studied the chemical constituents of the bark of the Ailanthus glandulosa tree and were able to isolate six canthinone-type alkaloids, also investigating the effectiveness of these constituents as anti-inflammatory [10].
Researchers synthesized copper oxide nanoparticles (ZnO-NPs) using the aqueous extract from the fruits of the Ailanthus glandulosa tree, then studied the effect of secondary copper oxide on some bacteria and concluded that this compound can be used as an antibacterial agent [11]. The researchers showed that the methanolic extracts of the leaves have the highest content of total phenols and have high antioxidant properties. They found that the acetone, methanol and dichloromethane extracts were very effective against Gram-positive bacteria but not against Gram-negative bacterial strains [12]. Researchers studied the chemical composition of Ailanthus glandulosa extract with chloroform as a solvent and demonstrated its toxic effect on whiteflies [13].
Plant extracts are currently a broad field for study due to their diversity and different uses. The aim of the research was therefore to: search for a new method to isolate the constituents of the leaves of Ailanthus glandulosa using a new solvent not previously used in reference studies, isopropanol, and investigate the chemical structures of the isolated compounds using GC/MS.

General information
The required chromatograms were obtained with a gas chromatography coupled GC/MS instrument of the SHIMADZU type, model GCMS-QP2010 plus, using a capillary column of the type (TRB-WAX) with dimensions (length 60 m * diameter 0.25 mm * thickness 0.25 μm) and a carrier gas containing helium with a purity of 99.9999. %. The mass spectra were recorded from m/z 15 to m/z 450.

Extract
The leaves of Ailanthus glandulosa were collected in June of 2021 in Lattakia Governorate, Syria. The leaves were thoroughly cleaned of dust and suspended impurities, dried in a well-ventilated, shaded place at room temperature (20-25 • C) for about one month, then crushed to obtain the required fineness.
The extract was obtained using a Soxhlet apparatus by adding 200 g of the crushed dried leaves into the thimble and then adding 1000 ml of isopropanol into the 2000 ml capacity flask of the apparatus. The extraction process took 72 h, after which the extract was concentrated using a rotary evaporator. A green, viscous extract was obtained at a temperature of 40 • C to a volume of 5 ml. Separation of the extract by column chromatography with the solvents (petroleum ether, dichloromethane, chloroform, methanol), yielding four eluates. The solvents of the four eluates were evaporated with a rotary evaporator at a temperature of 40 • C.For the first time, a new method was used to separate and isolate a number of chemical compounds from the leaves of Ailanthus glandulosa. The product was dissolved with methanol and then chloroform was added to form a precipitate in the form of a mixture of a white and a brown precipitate. The precipitate formed was dissolved again with methanol, then chloroform was added to the solution and a white precipitate and a brown filtrate were formed. The filtrate was treated with the following solvents: 2-methylbutane, pentane, chloroform, methanol, hexane, dichloromethane and diethyl ether, and a series of samples was obtained. 3. Chloroformeluate (C): Eluate C was treated as follows: a Using 2-methylbutane and chloroform, sample C 1 was obtained. b Using methanol and pentane, both samples C 2 were obtained. c Using methanol and dichloromethane, sample C 3 was obtained. d Samples C m and C n were isolated from sample C 2 using diethyl ether and methanol. e Sample C 3t was isolated using methanol and ethyl ether.

Organic feed analysis
Analysis was performed by injecting 2 μl of each sample into a device GC-MS at a flow rate of 1 ml/min. The solvent used was chloroform. Then the injector temperature was set at 250C ͦ , ionization source temperature at 225C ͦ and the quadrupole temperature at 130-150C ͦ . The thermal programme started at 200C ͦ , the temperature of the oven column, and this temperature was maintained for 1 min (1 min), then it was increased to 270C ͦ at a rate of 5C ͦ per minute, and this temperature was maintained for 35 min, and the mass spectra were recorded from 15 m\z to 450 m\z. The chemical compounds present in each pathway of the Ailanthus glandulosa leaf extract were determined by comparing the mass spectra generated for each peak with the mass spectra of the compounds in the HP Mass Sperliral Library Nist Wiley. The fragmentation mass spectra of some compounds were examined to confirm their structures based on the fragmentation mechanism shown in the recorded diagrams.

Results and discussion
The weight of the resulting isopropanolic extract was (4 g). This extract was treated with some solvents and separated into eluates containing fewer compounds by column chromatography. This makes it easier for us to examine the chromatograms of these eluates and accurately identify the components of the extract.

S.Y. Saad
The GC/MS chromatogram of sample B 1 (Fig. 2) shows the presence (99.98%) of a single compound, 2,4-diphenyl-4-methyl-1-pentene. The GC/MS chromatogram of sample B 2 (Fig. 3.) also shows the presence of a single compound, 1,3,3-trimethyl-1-phenylindane (98.01%), indicating good separation in column chromatography. The GC/MS chromatogram of sample (B 3 ) (Fig. 4) shows the presence of five ester compounds accounting for (97.87%) of the total sample. The compound with the highest percentage in the sample is 1,2-benzenedicarboxylic acid diisooctyl ester (41.14%), and the compound with the lowest percentage is di-ethylhexyl   The chemical structure of some compounds was confirmed by mass spectrometry. Table 1 shows the chemical constituents of the samples (B 1 , B 2 , B 3 ) of dichloromethaneluate. Fig. 5 shows the formulae of some chemical compounds contained in the samples of eluate B.
It was found that there are two aromatic compounds and three compounds belonging to the group of phthalic acid derivatives. These compounds (phthalates) may be of plant origin or due to environmental pollution, as we found them in the isopropanolic extract from the leaves of the Ailanthus glandulosa tree.
In the chromatograms of samples B 2 and B 3 (Figs. 3 and 4), small peaks were observed that were not detected by the instrument. From the GC/MS chromatogram of sample (C 1 ) (Fig. 6), it can be seen that six aldehydes, ketones and alcohols (98.12%) of the total sample are present. The compound with the highest percentage in the sample is 2-methyl-Z,Z-3,13-octadecadienol (27.10%), while the compound 2-methylundecanal has the lowest percentage (5.15%).
From the GC/MS chromatogram of sample (C 2 ) (Fig. 7), it can be seen that the six compounds are carboxylic acids, accounting for 99.75% of the total number of sample (C 2 ). The compound with the highest percentage in the sample is: tetradecanoic acid (C 2a ) (29.01%), while the compound heptadecanoic acid (C 2c ) has the lowest percentage (5.10%). Two compounds were isolated from the sample (C 2 ), namely (C m and C n ).
From the GC/MS chromatogram of sample C m (Fig. 8), it can be seen that it is a single compound. After investigating the fragmentation mechanism by mass spectrometry, the compound was found to be 2-naphthoxyacetic acid (C m ). The GC/MS chromatogram of sample C n (Fig. 9) shows that it is also a single compound. After investigating the fragmentation mechanism by mass spectrometry, the compound was found to be tridecanoic acid (Cn).
The GC/MS chromatogram of sample (C 3 ) (Fig. 10) shows the presence of five silicon compounds accounting for 97.38% of the total

S.Y. Saad
sample, the compound with the highest percentage being tetradecamethyl cycloheptasiloxane (60.87%). It was also found that there are other compounds with lower percentages than those mentioned above, the most important of which is: octadecamethyl cyclononasiloxane (24.98%). Only one compound C 3t , was isolated from sample C 3 . It is noted from the GC/MS chromatogram of sample C 3t (Fig. 11.) that there is only one compound, and it was found after a mechanical study. Fragmentation by mass spectrometry revealed that the compound is hexadecamethyl cyclooctasiloxane. Table 2 shows the chemical composition of the samples (C 1 , C 2 , C 3 ) from the chloroform eluate. Fig. 12 Shows the formulae of some chemical compounds in the samples of eluate C. There are small peaks that are not detected by the instrument. 4.The chemical compounds in the methanol eluate (D): The eluate (D) was treated with the following solvents: Methanol and Chloroform, and samples (D 1 -D 7 ) were obtained. Fig. 13 Shows the GC/MS chromatogram of sample (D 1 ), the presence of two compounds representing 89.95% of the total sample, the compound with the highest percentage in the extract is: 2-(2-ethoxyethoxy) -phosphate-ethanol (55.32%). The GC/MS chromatogram of the sample (D 2 ) (Fig. 14.) shows that one compound is 2,6-bis(1,1-dimethylethyl)-4-ethylphenol. As can be seen from the GC/MS chromatogram of sample (D 3 ) (Fig. 15.), the only compound is 2,5-di-tert-butylnitrobenzene, which accounts for 99.81% of the total sample. From the GC/MS chromatogram of sample (D 4 ) (Fig. 16), it can be seen that there is also a single compound, 2-allyl-2methyl-1,3-cyclopentanedione, accounting for (98.34%) of the total amount of the above sample. As can be seen from the GC/MS chromatogram of sample (D 5 ) (Fig. 17), the single compound is 2-ethylhexyl chloroformate.
The GC/MS chromatogram of sample (D 6 ) in Fig. 18 shows that the single compound is 5-hexyl-2-furaldehyde with a percentage of (99.14%). Fig. 19: The GC/MS chromatogram of sample (D 7 ) shows the presence of a single compound, 16-nitrobicyclo [10.4.0] hexadecan-1-ol-13-one, with a percentage of (99.98%). Table 3 shows the chemical composition of the methanol eluate samples (D eluate). Fig. 20 Shows the formulas of some chemical compounds in methanol eluate. There are small peaks that are not recognised by the device.

Conclusions
First use of isopropanol as a solvent for extraction. This method of separation and isolation was used for the first time in this study and had not been used previously. This method allowed the isolation of a greater number of chemical compounds with less solvent consumption. The leaves of Ailanthus glandulosa contain effective constituents in good quantity, and the percentage of isopropanol extract was 9% of the weight of the sample. The main constituents of the extract were determined and these compounds were found to be esters, aldehydes, ketones, alcohols, carboxylic acids and a number of silicone compounds. There will be a study of the effect of the isolated components on a number of bacteria and fungi. Taking care of this tree to benefit from its important components in many industrial fields.

Author contribution statement
Sawsan Saad: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Data availability statement
Data included in article/supp. Material/referenced in article.

Additional information
There is supplemental content related to this article.