Uncovering the Chemical Composition and Biological Potentials of Bupleurum lancifolium Hornem. from Jordan

The current study was designed to uncover the chemistry and bioactivity potentials of Bupleurum lancifolium growing wild in Jordan. In this context, the fresh aerial parts obtained from the plant material were subjected to hydrodistillation followed by GC/MS analysis. The main components of the HDEO were γ-patchoulene (23.79%), β-dihydro agarofuran (23.50%), α-guaiene (14.11%), and valencene (13.28%). Moreover, the crude thanolic extract was partitioned to afford two main major fractions, the aqueous methanol (BLM) and butanol (BLB). Phytochemical investigation of both fractions, using conventional chromatographic techniques followed by careful inspection of the spectral data for the isolated compounds (NMR, IR, and UV-Vis), resulted in the characterization of five known compounds, including α-spinasteryl (M1), ethyl arachidate (M2), ethyl myristate (M3), quercetin-3-O-β-d-glucopyranosyl-(1-4”)-α-L-rhamnopyranosyl (B1), and isorhamnetin-3-O-β-d-glucopyranosyl-(1-4”)-α-L-rhamnopyranosyl (B2). The TPC, TFC, and antioxidant activity testing of both fractions and HDEO revealed an interesting ABTS scavenging potential of the BLB fraction compared to the employed positive controls, which is in total agreement with its high TP and TF contents. Cytotoxic evaluation tests revealed that BLM had interesting cytotoxic effects on the normal breast cell line MDA-MB-231 (ATCC–HTB-26) and the normal dermal fibroblast (ATCC® PCS-201-012) and normal African green monkey kidney Vero (ATCC-CCL-81) cell lines. Despite both the BLB and BLM fractions showing interesting AChE inhibition activities (IC50 = 217.9 ± 5.3 µg/mL and 139.1 ± 5.6 µg/mL, respectively), the HDEO revealed an interestingly high AChE inhibition power (43.8 ± 2.7 µg/mL) that far exceeds the one observed for galanthamine (91.4 ± 5.2 µg/mL). The HDEO, BLM, and BLB exhbitied no interesting antimicrobial activity against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa.


Introduction
Umbelliferae (Apiaceae) family, famously known as the carrot family, is the 16th largest family of flowering plants, with more than 3540 species belonging to 446 genera [1,2].Aromatic flowering plants belonging to this family include economically important foods and spices, like caraway, anise, fennel, coriander, cumin, carrot, and many others.Most umbelliferae plants are annual, biennial, or perennial herbs, and less frequently shrubs or trees, that are known to be distributed worldwide but mostly in temperate climates [1].Among the different species belonging to this plant family, about 250 species have been reported as medicinally significant apiaceous plants utilized for ages in traditional medicine for treating an assortment of illnesses [3].
Several Bupleurum species, either alone or in combination with additional components, have been included in many pharmaceutical preparations and prescribed for the treatment of different ailments, including the common cold [5], inflammation [6], hepatitis [7], cancer [8], and fever associated with malaria [8].Additionally, Bupleurum species have been used as analgesics to alleviate persistent abdominal pain in the hypochondriac area of the chest, to prevent amenorrhea, and to treat and protect against chronic hepatitis, nephrotic syndrome, and autoimmune illnesses [9].Moreover, other uses include the treatment of diabetes, vertigo, vomiting, dry throat, and cholecystitis, as well as the enhancement of wound healing and deafness [10].The high concentration of polyacetylenes, which have very neurotoxic effects, is thought to be the cause of the poisonous nature of some of this genus's dangerous species [11].
Phytochemical investigation of several Bupleurum species has resulted in the identification of several classes of secondary metabolites, including flavonoids, coumarins, lignans, triterpenes saikosaponins, and polyacetylenes [12], in addition to the occurrence of free fatty acids, like pinellic acid, angelic acid, petrosylic acid, and lignoceric acid [13].These bioactive metabolites can operate as key substances in the therapy or avoidance of several severe diseases [14,15].
Bupleurum lancifolium Hornem. is an annual plant; the flowers are bisexual, with yellowish simple leaves having long and slender shapes (Figure 1).The plant has been reported to grow wild in many countries and regions, like Algeria, Cyprus, Egypt, Greece, Iran, Iraq, Kuwait, Lebanon-Syria, Libya, Madeira, Morocco, Palestine, Sinai, Spain, Tunisia, Turkey, Turkmenistan, and Western Sahara.The plant is reported to have anti-inflammatory, antitussive, hepatoprotective, anti-ulcer and immunomodulatory, antispasmodic, diaphoretic, antioxidant, and antimicrobial activities [16].Previous phytochemical investigations on the leaves of B. lancifolium resulted in the isolation of two triterpenoid saponins (3-O-[α-Lrhamnopyranosyl (1→4)-β-D-glucopyranosyl] echinocystic acid 28-O-β-D-glucopyranosyl ester and 3-O-[α-L-rhamnopyranosyl (1→4)-β-D-glucopyranosyl] oleanolic acid 28-O-β-D-glucopyranosyl ester) and two other flavonoids, including isorhamnetin 3-rutinoside and rutin [17].In Turkey, Saraço glu et al. (2012) investigated the chemical composition of the essential oil obtained from different organs of several Bupleurum species [18].The results revealed spathulenol and α-pinene as the primary constituents of the oil obtained from the flowers.Hexacosane and pentacosane dominated the essential oil obtained from fruit oil, while the roots oil contained mainly hexadecanoic acid and heptacosane [18].Another study reported the fatty acid composition of the oils extracted from B. lancifolium collected from the central Anatolia region of Turkey [19], reporting oleic acid, linoleic acid, and linolenic acids as the main components of the oils [19].Moreover, hexane extract obtained from B. lancifolium leaves and seeds extract contained ω-3, ω-6, palmitic acid, and γ-linolenic acid and is considered an important source of ω-3 and ω-6 compounds among several Bupleurum species [20].The main goal of the present study was to uncover the chemistry of B. lancifolium through investigating the composition of its hydrodistilled essential oil (HDEO) and aqueous methanol (BLM) and butanol (BLB) fractions.Furthermore, the two fractions (BLM and BLB) were assayed for their total phenolic contents (TPCs) and total flavonoid contents (TFCs).Both fractions, along with the HDEO, were tested for their antioxidant activity (DPPH and ABTS), antibacterial potentials (against a set of Gram-positive and Gramnegative bacteria), cytotoxic activity, and neuroprotective effects.

Chemical Constituents of the Hydrodistilled Essential Oil (HDEO)
The chemical composition of the HDEO obtained from fresh aerial parts of B. lancifolium was analyzed by GC-MS, and the results are shown in Table 1, while Figure S1 displays the obtained GC/MS chromatogram.A total of 48 different components, amounting to 99.76% of the total composition, were identified in this HDEO.The main goal of the present study was to uncover the chemistry of B. lancifolium through investigating the composition of its hydrodistilled essential oil (HDEO) and aqueous methanol (BLM) and butanol (BLB) fractions.Furthermore, the two fractions (BLM and BLB) were assayed for their total phenolic contents (TPCs) and total flavonoid contents (TFCs).Both fractions, along with the HDEO, were tested for their antioxidant activity (DPPH and ABTS), antibacterial potentials (against a set of Gram-positive and Gram-negative bacteria), cytotoxic activity, and neuroprotective effects.

Total Flavonoid (TFC) and Phenol (TPC) Contents and Antioxidant Activity
The results of the TPC, TFC, and antioxidant activity determined for the two fractions (BLB and BLM) are shown in Table 2.The obtained data clearly indicate that BLB had the highest TPC and TFC (849.46 ± 4.37 mg/g gallic acid equivalents; 405.23 ± 1.47 mg/g quercetin equivalents) compared to the BLM fraction.The DPPH • and ABTS •+ scavenging activity of the crude extracts and essential oil were dose dependent and increased with an increasing concentration (Figure S21.As shown in Table 2, both the BLB and BLM fractions showed good DPPH • radical scavenging powers ((IC50 = 8.0 ± 0.79 and 11.3 ± 0.20 µg/mL, respectively), which were less than that observed for HDEO (45.4 ± 0.50 µg/mL) when compared with the two positive controls ascorbic acid and α-tocopherol (1.58 ± 0.035 and 1.79 ± 0.01 µg/mL, respectively).The BLB fraction had the highest ABTS •+ scavenging potential (10.0 ± 0.54 µg/mL) compared to the BLM fraction (18.0 ± 0.35 µg/mL) and HDEO (41.0 ± 0.70 µg/mL).The observed activity for both fractions could be attributed to their TP and TF contents [21,22].Despite the fact that no phenolic compounds were detected in the GC/MS analysis of the HDEO, the moderate antioxidant potential recorded for the essential oil could be attributed to its terpenoidal content.The main components detected in the HDEO, including γ-patchoulene, 7-epi-α-selinene, β-dihydro agarofuran, and αguaiene, are known for their antioxidant properties [23,24].

Total Flavonoid (TFC) and Phenol (TPC) Contents and Antioxidant Activity
The results of the TPC, TFC, and antioxidant activity determined for the two fractions (BLB and BLM) are shown in Table 2.The obtained data clearly indicate that BLB had the highest TPC and TFC (849.46 ± 4.37 mg/g gallic acid equivalents; 405.23 ± 1.47 mg/g quercetin equivalents) compared to the BLM fraction.The DPPH • and ABTS •+ scavenging activity of the crude extracts and essential oil were dose dependent and increased with an increasing concentration (Figure S21.As shown in Table 2, both the BLB and BLM fractions showed good DPPH • radical scavenging powers ((IC 50 = 8.0 ± 0.79 and 11.3 ± 0.20 µg/mL, respectively), which were less than that observed for HDEO (45.4 ± 0.50 µg/mL) when compared with the two positive controls ascorbic acid and α-tocopherol (1.58 ± 0.035 and 1.79 ± 0.01 µg/mL, respectively).The BLB fraction had the highest ABTS •+ scavenging potential (10.0 ± 0.54 µg/mL) compared to the BLM fraction (18.0 ± 0.35 µg/mL) and HDEO (41.0 ± 0.70 µg/mL).The observed activity for both fractions could be attributed to their TP and TF contents [21,22].Despite the fact that no phenolic compounds were detected in the GC/MS analysis of the HDEO, the moderate antioxidant potential recorded for the essential oil could be attributed to its terpenoidal content.The main components detected in the HDEO, including γ-patchoulene, 7-epi-α-selinene, β-dihydro agarofuran, and α-guaiene, are known for their antioxidant properties [23,24].

Antibacterial, AChE Inhibition, and Cytotoxic Activity Assays
The antibacterial effects of the HDEO and the BLM and BLB fractions were evaluated against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa.The tested fractions and EO showed no antibacterial potentials at a 2 mg/mL concentration level and, accordingly, were considered inactive against all tested bacterial strains.However, the BLM fraction showed potent cytotoxic activity against the MDA-MB-231 breast cancer cell line at concentrations lower than that effective in inhibiting proliferation of both the normal fibroblast and Vero cell lines with the IC 50 (44.7 ± 1.3, 50.8 ± 2.1, and 106.9 ± 2.5 µg/mL, respectively) (Table 3).It is noteworthy that, although both the BLB and HDEO were ineffective against the tested cell lines, both extracts, as well as the BLM, revealed an ability to inhibit the activity of the AChE enzyme and, thereby, have potential roles in improving the cholinergic synapsis.The anti-AChE activities of the BLB, BLM, and HDEO were concentration dependent.Interestingly, the BLB and BLM displayed moderately weak AChE inhibition powers when compared to the positive control galantamine (IC 50 = 217.9± 5.3 and 139.1 ± 5.6 vs. 6.4 ± 2.1 µg/mL, respectively).The HDEO showed a stronger AChE inhibition potency compared to the two other fractions (IC 50 = 43.8± 2.7 µg/mL), which was three-to five-fold more effective than the BLB and BLM (Table 4).The present study's findings coincide with the literature on the medical applications of different Bupleurum species [5][6][7]15,16,[18][19][20].The inactivities of the tested BLM, BLB, and HDEO toward all tested bacterial strains have been previously reported by several studies for some Bupleurum species [18], including B. lancifolium; its essential oil [19] and crude extract [25] were devoid of activities against both Gram-negative and -positive bacteria.Intriguingly, the cytotoxic and anticholinergic activities of the BLM crude extract could be attributed to its contents of fatty acids and sterols (i.e., α-spinasteryl).Their activities were attributed to their lipophilic characteristic, which acts directly on biomembranes and affects membrane permeability, deteriorates plasma membrane, and might dissipate mitochondrial membrane potential [16,26,27].
Alpha-spinasterol has demonstrated potent cytotoxic activity on human ovarian, cervical Hela, and colon CACO-2 cell lines [28,29].Furthermore, this compound was also reported to cause inhibition in the growth of breast MDA-MB-231 and MCF-7 and ovarian SKOV-3 cell lines, acting mainly as an anti-estrogenic compound and possibly exerting its effect by binding to ER receptors and, thus, causing ER + MCF-7 cells to be more sensitive to α-spinasterol, caused by overexpression of p53 and downregulation in the cell cycle control Cdk4, leading to G0-G1 cell cycle arrest [30].Moreover, the ability of BLM to antagonize the AChE can also be correlated with the presence of α-spinasterol as a major component in this fraction, which is in total agreement with a previous report on its AChE inhibitory effect (IC 50 = 44.19± 2.59 µg/mL) [31].
However, the documented anticholinergic activity of HDEO herein could be attributed to its high sesquiterpenes content.The β-dihydro agarofuran derivatives obtained from the seeds of Maytenus disticha and M. magellanica inhibited AChE (IC 50 = 17.0 ± 0.016 and 740.0 ± 0.045 µM, respectively) [32].β-Dihydro agarofuran derivatives in Chilean Celastraceae extract caused an interesting AChE inhibitory effect (IC 50 values ranging from 120 ± 0.003 to 740.0 ± 0.035 µM) [33].The activity of this compound is attributed to its ability to form hydrogen bonds with the peripheral anionic site (PSA) at the entrance of the enzyme.Furthermore, an α-guaiene-rich extract obtained from Xylocarpus moluccensis roots showed anti-AchE potency (IC 50 = 21 µg/mL) [34].On the other hand, the presence of flavonoid glycosides like quercetin-3-O-β-D-glucopyranosyl-(1-4")-α-L-rhamnopyranosyl and isorhamnetin-3-O-β-D-glucopyranosyl-(1-4")-α-L-rhamnopyranosyl), as well as isorhamnetin derivatives, could account for the observed moderate AChE inhibitory effects of BLB fraction.Previously, isorhamnetin was reported to cause instability in Aβ aggregate neuroblastoma SH-SY5Y cells [27], and enhance synaptic plasticity and neurogenesis in the prefrontal cortex and hippocampus in scopolamine-induced amnesia mice model by improving the level of brain-derived neurotrophic factor (BDNF) [35].Moreover, the interaction between the active site of the AChE enzyme and the flavonol skeleton of both the quercetin and isorhamnetin derivatives decreases the activity of AChE [36], with the quercetin derivative being more potent than the isorhamnetin analogs.

General
1 H-NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer with TMS as an internal standard. 13C-NMR spectra were recorded at 125 MHz.High-resolution mass spectra (HRESIMS) were acquired by electrospray ionization with the positive-mode technique using a Bruker APEX-4 Mass spectrometer.UV-Vis spectra were recorded on a Shimadzu UV-1800 UV/Visible Scanning Spectrophotometer.TLC was performed on silica gel 60 GF254 precoated glass plates (0.25 or 0.50 mm in thickness, Macherey-Nagel).The compounds were visualized under UV light or spraying with sulfuric acid-anisaldehyde spraying reagent followed by heating.Analysis of the HDEO's constituents was performed on an Agilent 6890 series II-5973 GC-MS spectrometer interfaced with an HP chemstation.

Hydrodistillation of Essential Oil
Fresh aerial parts of B. lancifolium (200 g) were minced and suspended in distilled water (150 mL) and then subjected to hydrodistillation with a Clevenger apparatus for 3 h [37,38].The obtained oil was separated by extraction with diethyl ether (2.0 mL) twice.After evaporation of the diethyl ether, the resulting oil was dissolved in GC-grade n-hexane, dried over anhydrous sodium sulfate, and then stored in amber glass vials at 4-6 ºC.
The identification of the separated essential oil components was achieved by comparing their calculated Kovats retention (KI) to (C 8 -C 20 ) n-alkanes values with a column of identical polarity and under the same chromatographic conditions, as well as matching their recorded mass spectra with those listed in the built-in libraries' spectra (NIST, Gaithersburg, MD, USA and Wiley Co., Hoboken, NJ, USA).The principal components of the extracts were further identified by injecting authentic standard reference compounds under the same chromatographic conditions and from the literature [39].

Extraction and Isolation
The plant material was dried and crushed to a fine powder (8.0 kg) and then defatted for 10 days at room temperature using petroleum ether (20 L).Then, the defatted plant material was extracted with ethanol at room temperature (5 times, 7 days each).The combined ethanolic extract was concentrated under reduced pressure by evaporation, and the resulting alcoholic residue (700 g) was partitioned according to the procedure described in the literature [40] to obtain the aqueous methanol (BLM; 73.3 g) and butanol (BLB; 184.73 g) fractions.
The aqueous methanol fraction (BLM; 73.3 g) was adsorbed on 100.91 g of mesh silica gel and then chromatographed in a column (45 × 6 cm, 500 g mesh silica gel) packed in hexane and eluted with a gradient hexane/ethyl acetate mixture of increasing polarity A total of 177 fractions were collected (250 mL each) and then consolidated into 6 main subfractions (BLM-I-BLM-VI) based on their TLC behavior.Isolation and purification from these collective subfractions were then achieved by a combination of CC and TLC utilizing proper solvent systems.Three compounds (M1, M2, and M3) were isolated from the BLM fraction.
Similarly, the butanol fraction (BLB; 184.73 g) was adsorbed on 200.42 g mesh silica gel and then subjected to chromatography in a column (40 × 7 cm, 800 g mesh silica) packed in chloroform (CHCl 3 ) and eluted with a gradient mixture of CHCl 3 :MeOH of increasing polarity.A total of 213 fractions (250 mL each) were collected and then grouped into 5 major subfractions (BLB-I-BLB-V) according to their TLC behavior.Isolation and purification from these collective subfractions were then achieved by a combination of CC and TLC or a suitable solvent.This whole process resulted in the isolation and characterization of 2 compounds of the butanol fraction.

Determination of Total Flavonoid (TFC) and Phenol (TPC) Contents
Both the BLM and BLB fractions were tested for their TFC using Folin-Ciocalteu assay methods according to the procedure described in [37,38] with slight modification.Briefly, a 1.0 mL aliquot of the stock solution prepared from each fraction/EO (1 mg/mL) was diluted in 4.0 mL distilled water; then, 0.30 mL sodium nitrite solution (5% NaNO 2 , w/v) was added to a 10.0 mL volumetric flask.After 5 min, 0.30 mL of aluminum chloride solution (10% AlCl 3 , w/v) was added.The resulting solution was incubated for further 6 min and then 2.0 mL of 1.0 M NaOH solution was added to the mixture.The volume of the final solution was adjusted to 10.0 mL with distilled water.After 15 min, the absorbance was measured at 510 nm.Methanol was used as a blank.The TFC is expressed in mg quercetin/g of dry extract.
The TPCs for the BLB and BLM fractions were determined by aluminum chloride assay, as described previously [37,38].Briefly, 0.5 mL aliquot of each fraction stock solution (1 mg/mL) was treated with 2.5 mL of Folin-Ciocalteu reagent (2N) (diluted ten-fold) and 2 mL of Na 2 CO 3 (75 g/L).The mixture was kept at room temperature for 15 min; then, the absorbance was recorded at 765 nm.Methanol was used as a blank solution.The TPC is reported in mg Gallic acid/g of dry extract.

Determination of Antioxidant Activity
The antioxidant activity of the two fractions (BLB and BLM) and the HDEO was evaluated by the DPPH • and ABTS •+ assay methods, as described previously [37,38].Ascorbic acid and α-tocopherol were used as positive controls (Figure S21).

Antibacterial Activity
The antibacterial activities of the BLB, BLM, and HDEO were determined as described in the literature [45,46], following the Clinical and Laboratory Standards Institute's guidelines (CLSI, 2012), using the agar diffusion test.Several concentrations of the fractions/EOs (300 µg/disc, 500 µg/disc, 1 mg/disc, and 2 mg/disc) were applied on a surface of 6 mm sterile, blank discs placed on the top of Müller Hinton agar plates containing 10 6 cells/mL of the tested bacterial strains.The included bacterial strains were Gram-positive (B.cereus ATCC 11778; B. subtilis ATCC 6633; and S. aureus ATCC 43300) and Gram-negative strains (E. coli ATCC 25922 and P. aeruginosa ATCC 13048).The tests were conducted in three independent experiments.

In Vitro Cytotoxicity Assay
The antiproliferative potencies of the BLB, BLM and HDEO were evaluated against three monolayer cell lines, namely, the human breast adenocarcinoma MDA-MB-231 (ATCC-

Figure 2 .
Figure 2. Main components detected in the HDEO of B. Lancifolium.Figure 2. Main components detected in the HDEO of B. Lancifolium.

Figure 2 .
Figure 2. Main components detected in the HDEO of B. Lancifolium.Figure 2. Main components detected in the HDEO of B. Lancifolium.

Figure 4 .
Figure 4.Chemical constituents of B. lancifolium isolated from the BLM and BLB fractions.

Figure 4 .
Figure 4.Chemical constituents of B. lancifolium isolated from the BLM and BLB fractions.

Table 1 .
Chemical constituents and their percentage of the composition in the HDEO obtained from aerial parts of B. lancifolium.

Table 1 .
Chemical constituents and their percentage of the composition in the HDEO obtained from aerial parts of B. lancifolium.
a KI = Kovats retention index experimentally calculated.b % Area of the peak.c MS: Identification by mass spectrum.d CoI: Co-injection with authentic compound. .
a KI = Kovats retention index experimentally calculated.b % Area of the peak.c MS: Identification by mass spectrum.d CoI: Co-injection with authentic compound.

Table 2 .
Results for the TPC (mg/g gallic acid equivalents) and TFC (mg/g quercetin equivalents) of the BLM and BLB fractions and the IC50 (µg/mL) of the antioxidant activities of the HDEO and BLM and BLB fractions obtained from B. lancifolium.

Table 2 .
Results for the TPC (mg/g gallic acid equivalents) and TFC (mg/g quercetin equivalents) of the BLM and BLB fractions and the IC 50 (µg/mL) of the antioxidant activities of the HDEO and BLM and BLB fractions obtained from B. lancifolium.

Table 3 .
Cytotoxic effects of extracts (BLB, BLM, and essential oil) and daunorubicin against the MDA-MB-231, fibroblast, and Vero cancer cell lines.
IC 50 values (µg/mL) with similar letters are not significantly different from each other based on the post hoc Tukey HSD test.Asterisks indicate statistically significant differences in the IC 50 values of the positive control to the tested samples (* p < 0.05).

Table 4 .
Acetylcholine esterase (AChE) enzyme % inhibition activities and IC 50 (µg/mL) evaluations of the BLB, BLM, and HDEO compared to the positive control galantamine.
dIC 50 values (µg/mL) with similar letters are not significantly different from each other based on the post hoc Tukey HSD test.