Phytochemical investigation using GC/MS analysis and evaluation of antimicrobial and cytotoxic activities of the lipoidal matter of leaves of Sophora secundiflora and Sophora tomentosa

This study aims at the investigation of the phytochemical composition, antimicrobial and cytotoxic activities of the lipoidal matter of leaves of S. secundiflora (Ortega) and S. tomentosa L. The saponifiable and unsaponifiable matter of S. secundiflora and S. tomentosa leaves were assessed using GC/MS analysis. Where, saponification of lipoidal matter of S. secundiflora and S. tomentosa leaves yielded 31.55%, 87.74% for unsaponifiable matter, and 19.66%, 38.70% for fatty acids methyl esters of both species, respectively. The dominant compounds in the unsaponifiable matter of S. secundiflora were β-amyrin acetate 55.20% and α-amyrin 9.73%. Whereas n-nonacosane 43.80% and 2methyltriacontane 11.94% were the main components in S. tomentosa. In the saponifiable fraction, the content of saturated fatty acids identified in S. tomentosa 58.37% is higher than S. secundiflora 29.0%, while the percentage of unsaturated fatty acids identified in S. secundiflora 62.67% is higher than S. tomentosa 34.51%. Methyl linolenate 36.62% and methyl palmitate 40.02% are the major compounds in S. secundiflora and S. tomentosa, respectively. The lipoidal matters were evaluated in vitro for cytotoxic activity towards HCT-116 carcinoma cell line using the MTT assay with an IC50 value of 97.00 and 38.76 μg/mL for S. secundiflora and S. tomentosa, respectively. Using the technique of agar well diffusion, the lipoidal matter of S. secundiflora and S. tomentosa displayed moderate antimicrobial activity at conc. of 50 mg/mL.


INTRODUCTION
Genus Sophora belongs to the family Fabaceae; comprises about 52 species [1]. This has a diverse array of pharmacological properties including cytotoxic, antimicrobial, antifungal, anti-diabetic, and anti-inflammatory and neuroprotective activities [2-4]. Sophora secundiflora (Ortega) Lag. ex DC [syn. Calia secundiflora (Ortega) Yakovlev] and reclassified as Dermatophyllum secundiflorum [5, 6] is a bushy plant that is spread throughout Africa, America, and Asia across southern Mexico [7]. Historically, the roots are used to treat inflammation and sore throat and as an antipyretic, analgesic, antidote, antitumor, antiparasitic, and diuretic as well [8,9]. Sophora tomentosa L. is a shrub found all over China, Tanzania, Sri Lanka, and Queensland. Traditionally, it has medicinal importance as a remedy for cholera, diarrhea, and stomach disorders, also antidote after eating poisonous fish and other marine animals [10,11]. Also, it was used for the treatment of hypertension in Taiwan folk medicine [12]. A myriad of active compounds including alkaloids, flavonoids, steroids, and triterpenoids compounds were isolated from the genus Sophora [13][14][15][16].
This study intended to identify and compare the lipoidal matter of leaves of S. secundiflora and S. tomentosa using GC/MS analysis to widen the range of phytochemicals and biological investigations that were carried on Sophora members and to evaluate their cytotoxic and antimicrobial activities. This, to the best of our knowledge, is the first study of the phytochemical composition and evaluation of antimicrobial and cytotoxic activities of the lipoidal matter of S. secundiflora and S. tomentosa leaves.

Plant material
Leaves of S. secundiflora were collected from El Zohreya Botanical Garden and leaves of S. tomentosa were collected from El Orman Botanical Garden, Giza, Egypt in December 2016. The taxonomic authentication was performed by the taxonomy specialist Terease Labib, Consultant of Plant Taxonomy at the Ministry of Agriculture, Egypt. The identity was ascertained by DNA profiling performed by the authors [17]. Samples of the plant material were placed at the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt with codes (PHG-P-SS-206) and (PHG-P-ST-207) for S. secundiflora and S. tomentosa, respectively.

Preparation of the lipoidal matter
The air-dried powder of leaves of S. secundiflora and S. tomentosa (130 g) were each independently exhaustively extracted with light petroleum ether (b.p. 60-80 °C) (3x250 mL) for 3 days. Both concentrates were evaporated individually under reduced pressure and produced petroleum ether extracts 4.12 g and 3.10 g (lipoidal matter), respectively [18].

Preparation of the unsaponifiable matter
The prepared lipoidal matter of both plants was individually saponified by refluxing with 50 mL of 30% alcoholic KOH for 3 h followed by distillation of the alcohol under reduced pressure and dilution with 100 mL distilled water. The aqueous solution was extracted with diethyl ether (5 x 100 mL) in a separating funnel several times till complete exhaustion then, washed several times with distilled water till complete free alkalinity, anhydrous Na 2 SO 4 used for dehydration. The extract was concentrated under reduced pressure to afford 1.30 g and 2.72 g of S. secundiflora and S. tomentosa unsaponifiable matter (USM), respectively. Both of them were kept in sealed containers for further investigation [19].

Isolation of free fatty acids
Upon extraction of the unsaponifiable material, the aqueous alkaline layer left was acidified with 10% HCl gradually and the liberated fatty acids were extracted with diethyl ether (5 x1 00 mL) till exhaustion and then washed with distilled water until free of acidity, anhydrous Na 2 SO 4 used for dehydration after that evaporated under reduced pressure to provide residue of total fatty acids 0.95 g and 1.38 g for S. secundiflora and S. tomentosa, respectively [19].

Preparation of fatty acid methyl esters
The free fatty acid fractions of both S. secundiflora and S. tomentosa were methylated by dissolving in 25 mL methanol, 2 mL concentrated H 2 SO 4 and each mixture was refluxed for 3 h to produce fatty acid methyl esters. The methanolic solution was evaporated; the residue was diluted with 100 mL of distilled water and then extracted with ether (5 x 100 mL). Each of the combined ethereal extracts was washed with distilled water until neutral to litmus paper, anhydrous Na 2 SO 4 used for dehydration, then evaporation under reduced pressure to provide fatty acid methyl esters (FAME) 0.81 g and 0.62 g for S. secundiflora and S. tomentosa. Both were kept in sealed vials for GC/MS analysis [20-22].

Cytotoxic activity
The cytotoxicity activity of the lipoidal matter of S. secundiflora and S. tomentosa was estimated using MTT assay against HCT-16 (human colon carcinoma cell line). The cell viability was expressed as a percentage of control and estimation of the concentration that induces 50% of maximum inhibition of cell proliferation (IC 50 ) from graphic plots of the dose-response curve for each concentration using Graphpad Prism software (San Diego, CA, USA) [31-35].

Antimicrobial activity
The lipoidal matter of S. secundiflora and S. tomentosa at a concentration of 50 mg/mL were evaluated for their antimicrobial activity using the agar well diffusion technique against the Gram-positive bacteria Bacillus subtilis (RCMB 015(1) NRRL B-543), Staphylococcus aureus (RCMB 010010) and the Gram-negative bacteria Escherichia coli (ATCC 25955), Pseudomonas aeruginosa (NCIB-9016). Also, fungal strains Candida albicans (ATCC-10231) and Aspergillus niger (RCMB 0020080) according to the National Committee of Clinical Laboratory Standards (NCCLS) [38, 39]. The positive antibacterial and antifungal activities were estimated by the presence of measurable zones of inhibition for bacteria after 24 h incubation period and for fungi after 48 h. Gentamycin (4 µg/mL) and Ketoconazole (100 µg/mL) were used as positive reference antibiotics and antifungal drugs, respectively.

GC/MS analysis
Petroleum ether extract saponification of S. secundiflora and S. tomentosa leaves yielded 31.55% and 87.74% for unsaponifiable matter (USM), while 19.66% and 38.70% for fatty acids methyl esters (FAME), respectively. The lipoidal matter of both species S. secundiflora and S. tomentosa were qualitatively and quantitatively analyzed using GC/MS technique. The results revealed the existence of 9 compounds in the unsaponifiable matter (USM) of S. secundiflora while, 19 compounds in S. tomentosa accounting for 84.68% and 92.97%, respectively (Table 1). In addition, a total of 15 and 31 compounds were specified in the saponifiable fraction of S. secundiflora and S. tomentosa accounting for 95.53% and 93.70%, respectively ( Table 2). The GC chromatograms are displayed in (Fig. 1. and  Fig. 2). The structures of the main identified compounds of the lipoidal matter of both plants are illustrated in (Fig. 3).      Total identified hydrocarbons in USM of S. secundiflora were 4 compounds, representing 7.11% of the total identified unsaponifiable compounds. Furthermore, 9 hydrocarbons were specified in S. tomentosa accounting for 67.37% of the total identified unsaponifiable compounds, mainly attributed to n-nonacosane accounting for 3.70% for S. secundiflora and 43.80% for S. tomentosa. Besides, 2-Methyltriacontane (C 31 H 64 ) a monomethyl-branched alkane [40] accounting for 2.67% for S. secundiflora and 11.94% for S. tomentosa.
The percentage of identified fatty acids in S. secundiflora was 29.0% and 62.67% for saturated fatty acids and unsaturated fatty acids, respectively. While in S. tomentosa the percentage was 58.37% and 34.51% for saturated fatty acids and unsaturated fatty acids, respectively. Results of GC/MS analysis of the FAME showed that the major compound is methyl palmitate accounting for 17.06% for S. secundiflora and 40.02% for S. tomentosa. In the saponifiable fraction of S. secundiflora, linolenic acid methyl ester 36.62% and methyl linolenate 22.12% were detected. Furthermore, oleic acid methyl ester 22.87% and methyl linolenate 8.94% are the major unsaturated fatty acids in S. tomentosa saponifiable fraction. In the saponifiable fraction of S. tomentosa, different saturated and unsaturated fatty acids were identified including oleic acid methyl ester 22.84%, stearic acid methyl ester 5.71%, myristic acid methyl ester 3.11%, eicosanoic acid methyl ester 2.07%, behenic acid methyl ester 1.41% and pentadecanoic acid methyl ester 1.26%.

Cytotoxic activity
The cytotoxicity of the lipoidal matter was evaluated using HCT-116. The IC 50 values are represented in (Fig. 4). The highest cytotoxic activity was observed for S. tomentosa with an IC 50 value of 38.76 μg/mL, while secundiflora with an IC 50 value of 97.0 μg/mL.

Antimicrobial activity
By using the agar well diffusion technique, the lipoidal matter of S. secundiflora and S. tomentosa leaves were screened for antimicrobial activity at conc. of 50 mg/mL against selected microbial strains. The average diameters of the growth inhibition zones are listed in (Table 3). The zone of inhibition diameter was used for estimation of the antimicrobial activity where inactive when the zone of inhibition diameter <9 mm. The partial activity was reported with a zone of inhibition diameter ranged from 9 to 12. While active when the zone of inhibition diameter range 13-18 mm and very active when the zone of inhibition diameter >18 mm [41]. Our results revealed that the lipoidal matter of S. secundiflora showed partial activity against B. subtilis, Staph. aureus and E. coli with inhibition zones of 10, 11, and 11 mm diameter, respectively. While the lipoidal matter of S. tomentosa was partially active against Staph. aureus and active against E. coli with inhibition zones of 9 and 14 mm diameter, respectively, and no activity was observed against B. subtilis. Both lipoidal matters showed no activity towards Klebsiella pneumonia, Candida albicans, and Aspergillus niger.

DISCUSSION
The GC/MS analysis of both lipoidal matters of S. secundiflora and S. tomentosa revealed the presence of bioactive components as phytol a cyclic diterpene and a member of branched-chain unsaturated alcohols with antioxidant activity related to antinociceptive activities [42]. αand β-amyrins are pentacyclic triterpenes with antioxidant, antimicrobial, anti-inflammatory, and anticancer properties [43, 44]. Linolenic acid C 18:3 is a polyunsaturated fatty acid called omega-3 fatty acid relative to its three double bonds. It is essential for all mammals; its consumption might reduce heart disease mortality and has a preventative effect against cardiovascular diseases [45,46].
A recent previous study by the authors concerning the GC/MS analysis of the essential oil of flowers of S. secundiflora and S. tomentosa. The study reported the prevalence of fatty acid methyl and ethyl esters accounting for 4.63% and 2.72% of the total components in S. secundiflora and S. tomentosa, respectively. In S. secundiflora essential oil the following fatty acids are detected; methyl and ethyl palmitate, linolenic acid methyl and ethyl ester, linoleic acid ethyl ester, myristic acid methyl, and ethyl ester and lauric acid methyl ester. While in S. tomentosa essential oil; methyl palmitate and methyl linolenate were identified [47].
The highest cytotoxic activity was observed for S. tomentosa with an IC 50 value of 38.76 μg/mL against HCT-116 that might be attributed to synergistic potentiation between plant components present in the lipoidal matter that may improve its biological effects [55]. Where 19 compounds were identified in the unsaponifiable matter of S. tomentosa accounting for 92.97%, and 31 compounds were identified in the saponifiable fraction of S. tomentosa accounting for 93.70%. According to our results, stigmasterol, sitosterol, and campesterol are basic phytosterols of plant cell membranes that are numerous in vegetable oils, nuts, seeds, and grains [56]. They are considered to have miscellaneous biological activities including antiinflammatory, anti-oxidant, and anti-carcinogenic activities, and also their capacity of cholesterollowering The antimicrobial activity would be assigned to the existence of phytosterol and fatty acids. Sterols are membrane lipophilic components playing a key role in its fluidity and have numerous biological activities [69]. Furthermore, linoleic acid and linolenic acid have been reported for their antibacterial activities against S. aureus and B. subtilis [70].

CONCLUSION
The present study indicated the existence of bioactive lipophilic compounds in S. secundiflora and S. tomentosa that make them a great source for natural health products. Hydrocarbons were the major components identified in S. tomentosa representing 67.37% of the total identified unsaponifiable compounds, mainly attributed to n-nonacosane accounting for 43.80%. While terpenoids were the major components identified in S. secundiflora representing 71.17% of USM, where, β-amyrin acetate 55.20% is the major compound. Methyl linolenate 36.62% is the major compound in the saponifiable fraction of S. secundiflora. While methyl palmitate 40.02% is the major compound in the saponifiable fraction of S. tomentosa. Antibacterial activity for both species was moderate and they didn't show an antifungal activity was observed on both tested fungal strains. Sophora tomentosa lipoidal matter showed higher cytotoxic activity towards HCT-116 with an IC 50 value of 38.76 μg/mL, while secundiflora with an IC 50 value of 97.0 μg/mL. Sophora secundiflora and S. tomentosa are worthy candidates for more comprehensive pharmacological and phytochemical studies owing to their prospect as a source of biologically active compounds.

Ethics approval and consent to participate
Not applicable

Consent to publish
Not applicable

Availability of data and materials
All data generated or analyzed during this study were included in the main manuscript

Competing interests
The authors declare that no competing interests exist

Funding statement
No funding source was received