GC/MS analysis of petroleum ether extract

The chemical constituents of the petroleum ether extract of both H and R were identified using GC/MS analysis. Table 1 (Figs. 1 and 2) showed that the petroleum ether extracts of H and R of A. caprinus contained 34 and 35 compounds, respectively, belonging to different classes of phytoconstituents, including hydrocarbons which constitute 53.5% and 55.17%, respectively, with n-undecane as main one (14.32% and 11.83%, respectively), aromatic components (34.92% and 35.20% ) in which 5-phenyl undecane is the main compound (4.34% and 3,49%, respectively), only one monoterpene hydrocarbon (p- menthane) in both H and R (2.35% and 1.69%). Sesquiterpene hydrocarbons are present only in H (1.77%) and absent in R, in addition to acids, which are absent in H and present in R (2.12%). To our knowledge, these data are reported for the first time for A. caprinus ssp. langarise, Table 2; Fig. 3 shows the percentage of the different compound classes of the petroleum ether extract of H and R. Considering other species, such as A. sieberi, a study revealed the presence of four compounds from the pet. ether fraction with N, N-dimethyl-1-Dodecanamine (42.36%), and butylated hydroxytoluene (35.96%) as major compounds detected in the GC/MS analysis constituting 78.32% of the total peak area [26]. B-sitosterol and ceryl alcohol were isolated from the unsaponifiable fraction of Astragalus cremophilos, and the fatty acids were studied by GLC [27].

Table 1 GC/MS data of petroleum ether extract of both H andFig. 1

GC chromatogram of petroleum ether extract of H

Fig. 2

GC chromatogram of petroleum ether extract of R

Table 2 Classes of Pet. ether ext of both H and RFig. 3

Percentage of compound classes present in both H and R

GLC analysis of unsaponifiable fraction

The unsaponifiable matter of both H and R was examined using GLC. The total ion chromatograms are displayed in Figs. 4 and 5 and the components are shown in Table 3. The GLC analysis of H revealed a combination of triterpenes, sterols, and hydrocarbons. The predominant hydrocarbon was C14 (32.98%), with the other hydrocarbons ranging from C11 to C30. Campesterol was the predominant sterol (1.62%), and only one triterpene (β- amyrin) was detected. The GLC analysis of R revealed the presence of a mixture consisting of sterols and hydrocarbons in which the predominant hydrocarbon is C25 (54.16%), with the other hydrocarbons ranging from C11 to C30. Campesterol was also the predominant sterol (5.67%).

Figure 6 Displays the hydrocarbon percentage of H and R.

Table 3 GLC data of Unsaponifiable fractions of both H and RGLC analysis of fatty acid methyl esters

The data of the GLC analysis of the fatty acid methyl esters of both H and R (Figs. 7 and 8) displayed nine fatty acids in H, accounting for 94.25 of the total acid percentage. The primary unsaturated fatty acids are linolenic acid (33.53%) and linoleic acid (15.15%), while the major saturated fatty acid is palmitic acid (24.90%). The results of R revealed the presence of eight fatty acids, representing 99.46% of the total. Linolenic acid was the main one (23.11%), followed by Linoleic acid (22.05%). The major saturated fatty acid was palmitic acid (21.03%), as shown in Table 4. Figure 9 shows the percentage of unsaturated fatty acids in H and R.

Fig. 4

GLC chromatogram of Unsaponifiable fraction of the H

Fig. 5

GLC chromatogram of Unsaponifiable fraction of the R

Fig. 6

Percentage of fatty acids of both H and R

Fig. 7

GLC chromatogram of fatty acid methyl esters of H

Fig. 8

GLC chromatogram of fatty acid methyl esters of R

According to Keskin and Kaçar [28], the Astragalus species have been found to contain significant amounts of palmitic (C16), linoleic (C18:2\omega-6), linolenic (C18:3\omega-3), and stearic acid (C18:0) in the roots and shoots. The fatty acid composition of Astragalus exscapus L. subsp. transsilvanicus roots were also studied by Szabo [29], and it was found that linoleic acid was the most abundant compound, followed by palmitic, oleic, and α-linolenic acids.

Table 4 GLC data of fatty acid methyl esters of H and RFig. 9

Percentage of unsaturated fatty acids in H and R

Identification of flavonoids Compound 1

luteolin, this compound was isolated as an amorphous yellowish powder, and it exhibited band-I in the UV spectrum with methanol at λmax = 347 nm, which proves its flavone nature. The presence of an ortho dihydroxy system was confirmed through AlCl3/HCl spectrum, where there is a hypthochromic shift (34 nm) in the band–I relative to AlCl3 spectrum. The MS spectrum gave M + at m/z = 286, corresponding to the molecular formula C15H10O6 [30].

Compound 2

After re-chromatography, cosmosiin (apigenin-7-O-glucoside) was isolated as a pale yellow powder. The UV spectra of the compound in methanol and different shift reagents confirmed its flavone nature, characterized by band-I at λmax = 330nm, and the absence of an ortho dihydroxy system. The acid hydrolysis of the compound resulted in the formation of apigenin as an aglycone and glucose as a sugar. The attachment of the sugar moiety at C-7 was confirmed through band-2 at λmax = 274 nm in the sodium acetate spectrum relative to band-2 at λmax = 267 nm in methanol. The mass spectrum of the compound gave M+ at m/z 432, which corresponds to the molecular formula C21H20O10. The 1H-NMR data confirmed the presence of apigenin as an aglycone and only one glucose moiety as a sugar, where signals were observed at δ = 6.39 (d, J = 2.4 Hz, H-6), 6.78 (d, J = 2.4 Hz, H-8), 6.82 (s, H-3), 6.90 (d, J = 9.1 Hz, H-3’, H-5’) and 7.88 (d, J = 9.1 Hz, H-2’, H-6’). The anomeric proton of the glucose appeared at 5.39 (d, J = 6.9 Hz, H-1”) and δ C at 99.87 (C-1”). The other data obtained were consistent with previously reported data [30].

Compound 3

cynaroside (luteolin-7-O-glucoside) was isolated from the butanol fraction after column chromatography and obtained as a yellowish amorphous powder. Its chromatographic behavior and acid hydrolysis substantiate its glycosidic nature with only one glucose moiety. The UV spectra showed absorption maxima of band–I in methanol at λmax = 348 nm, which proved its flavone nature; also, it established the absence of a free OH group at C-7 with no bathochromic shift in band-II (260 nm) of sodium acetate spectrum relative to methanol spectrum (256 nm). The EI-MS spectrum showed the molecular ion peak M+ as a small peak at m/z = 448, which fit the molecular formula C21H20O11 corresponding to luteolin-7-O-glucoside. The 1H-NMR data displayed signals at δ = 6.79 and 6.44 assigned for H-8 and H-6( J = 2.1 Hz), 6.79(s, 1H, H-3) indicates the flavone nature of the compound, 6.89 (1H, d, J = 8.2 Hz, H-5′), 7.40 (1H, d, J = 2.1 Hz, H-2′), 7.45 (1H, dd, J = 8.3, 2.1 Hz, H-6′); the anomeric proton of glucose was assigned at δ = 5.09 (1H, d, J = 7.5 Hz, H-1″) in addition to the rest of glucose protons at range of δ = 3.16–3.7, which means the presence of luteolin with glucose [30]. The chemical structure of isolated flavonoids is presented in Fig. 10.

Fig. 10

Chemical structure of isolated flavonoids

Antimicrobial activity

The growth absence of the microorganism (clear zone) and the diffusion of an antibiotic agent in the medium after 24 h for evaluation are related to the inhibitory zone in the well diffusion method. As indicated in Table 5, the investigation revealed that the subsequent extracts of both the H and R demonstrated differing degrees of activity against the strains that were examined, except for the Gram-negative bacteria Klebsiella pneumonia, the Gram-positive bacteria Streptococcus mutans, and the fungus Aspergillus niger.

Cytotoxic assay

The in vitro cytotoxicity of A. caprinus 80% MeOH extracts of H and R against HepG-2 and MCF-7 human carcinoma cell lines was evaluated using an MTT assay with Doxorubicin as a positive control. The obtained results are given in Table 6.

Table 5 Antimicrobial activity (inhibition zone in mm) of the successive extracts of both H and R of A. caprinusTable 6 The IC50 values of H and R methanol extracts using MTT assay