Fathi Mabrouki

New antiplasmodial alkaloids from Stephania rotunda

ETHNOPHARMACOLOGICAL RELEVANCE Stephania rotunda Lour. (Menispermaceae) is a creeper growing in many countries of Asia and commonly found in the mountainous areas of Cambodia. As a folk medicine, it has been mainly used for the treatment of fever and malaria. The pharmacological activity is mostly due to alkaloids. Thus the aim of this study is to isolate new bioactive alkaloids from Stephania rotunda and to evaluate their in vitro antiplasmodial activity. MATERIALS AND METHODS Alkaloids were isolated and identified from dichloromethane and aqueous extracts using a combination of flash chromatography, high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The purified compounds were tested for in vitro antiplasmodial activity on chloroquine-resistant W2 strain of Plasmodium falciparum. RESULTS A new aporphine alkaloid named vireakine (2) along with two known alkaloids stephanine (1) and pseudopalmatine (8), described for the first time in Stephania rotunda, and together five known alkaloids tetrahydropalmatine (3), xylopinine (4), roemerine (5), cepharanthine (6) and palmatine (7) were isolated and identified. The structure of the new alkaloid was established on the basis of 1D and 2D NMR experiments and mass spectrometry. The compounds were evaluated for their in vitro antiplasmodial and cytotoxic activities. All tested compounds showed significant antiplasmodial activities with IC(50) ranged from 1.2 μM to 52.3 μM with a good selectivity index for pseudopalmatine with IC(50) of 2.8 μM against W2 strain of Plasmodium falciparum and IC(50)>25 μM on K562S cells. CONCLUSIONS This study provides evidence to support the use of Stephania rotunda for the treatment of malaria and/or fever by the healers. Alkaloids of the tuber exhibited antiplasmodial activity and particularly cepharanthine and pseudopalmatine.

Chemical profiling of the tuber of Stephania cambodica Gagnep. (Menispermaceae) and analytical control by UHPLC-DAD

Abstract A new aporphine glycoside (1), named ‘angkorwatine’, and eight known alkaloids: oblongine (2), stepharine (3), asimilobine-β-d-glucopyranoside (4), isocorydine (5), tetrahydropalmatine (THP) (6), jatrorrhizine (7), palmatine (PAL) (8), and roemerine (ROE) (9) were simultaneously isolated from the tuber of Stephania cambodica. The development and validation of UHPLC-DAD method was carried out for the quantification of marker compounds (PAL, ROE, THP) of S. cambodica. In addition to good selectivity and linearity (r2 > 0.997), trueness, precision, and accuracy of the method did not exceed the acceptance limit of ±10% for ROE, THP and ±20% for PAL. Consequently, this method is able to provide accurate results between 1.39–4.18 μg/mL, 2.01–30.72 μg/mL, and 4.29–64.42 μg/mL for PAL, ROE, and THP, respectively. This study shows that the validated UHPLC method is a rapid, innovative and effective analytical approach to control quality of tubers of S. cambodica and to regulate the usage of this plant in traditional medicine.

HPLC Analysis of Stephania rotunda Extracts and Correlation with Antiplasmodial Activity

Stephania rotunda (Menispermaceae), a creeper commonly found in the mountainous areas of Cambodia, has been mainly used for the treatment of fever and malaria. Thus, the aim of this study is to investigate the chemical composition and antiplasmodial activity of different samples of S. rotunda and compare their antiplasmodial activity with their alkaloid content. Sixteen samples from different parts (roots, stem, and tuber) of S. rotunda were collected from four regions of Cambodia (Battambang, Pailin, Siem Reap, and Kampot). Reversed‐phase HPLC was used to determine the content of three bioactive alkaloids (cepharanthine, tetrahydropalmatine, and xylopinine). These three alkaloids have been found in all samples from Battambang and Pailin (samples I–IX), whereas only tetrahydropalmatine was present in samples from Siem Reap and Kampot (samples X–XVI). The analyzed extracts were evaluated for their antiplasmodial activity on W2 strain of Plasmodium falciparum. Among them, 13 extracts were significantly active with inhibitory concentration 50 (IC50) from 1.2 to 3.7 µg/mL and 2 extracts were moderately active (IC50 = 6.1 and 10 µg/mL, respectively), whereas sample XI was not active (IC50 = 19.6 µg/mL). A comparison between antiplasmodial activity and concentration of the three bioactive alkaloids in S. rotunda extracts has been realized. Copyright

Biologically Active Compounds from Lepidium campestre and Pulp from Lemon-Juice Production

In continuation of the search for possible new sources of biologically active compounds, we studied the aerial parts of Lepidium campestre (L.) W.T.Aiton of the family Cruciferae Juss. that was growing in Azerbaijan [1] and fruit of Citrus limon Burm. of the family Rutaceae Juss. [2, 3]. Aerial parts of L. campestre were collected during full flowering in the middle of July 2012 in the vicinity of Kubinsky District, Azerbaijan Republic. Pulp was produced during lemon-juice production at facilities in Lenkaransky Districts in autumn 2014. Air-dried ground aerial parts (1.0 kg) of L. campestre were extracted with EtOH (95%). The extracts were evaporated to 150–200 mL, diluted with H2O (150 mL), and evaporated to an aqueous residue that was worked up sequentially with CHCl3, EtOAc:hexane, and EtOAc. The EtOAc–hexane extract afforded compound 1, C15H10O6, mp 275–277°C (EtOH), tetraacetate mp 182–184°C (CHCl3–MeOH). Kaempferol (3,5,7,4 -tetrahydroxyflavone) (1). 1Í NMR spectrum (600 MHz, DMSO-d6, ppm, J/Hz): 6.18 (1Í, d, Í-6), 6.39 (1Í, d, Í-8), 8.08 (1Í, d, Í-2 ), 6.90 (2Í, d, Í-3 , 5 ), 8.08 (1Í, d, Í-6 ). 13C NMR spectrum (150 MHz, DMSO-d6, ppm): 148.1 (Ñ-2), 137.1 (Ñ-3), 177.4 (Ñ-4), 162.5 (Ñ-5), 99.3 (Ñ-6), 165.6 (Ñ-7), 94.5 (Ñ-8), 158.3 (Ñ-9), 104.5 (Ñ-10), 123.7 (Ñ-1 ), 130.7 (Ñ-2 ), 116.2 (Ñ-3 ), 160.0 (Ñ-4 ), 116.2 (Ñ-5 ), 130.7 (Ñ-6 ) [4]. Compound 2 was isolated from the EtOAc extract and was a flavonoid [5] of formula C26H28O14, mp 210–212°C (EtOH). Acid hydrolysis cleaved 2 into kaempferol (48%), L-arabinose, and L-rhamnose. The attachment site of the sugars to the aglycon was determined as before [6]. Alkaline hydrolysis of 2 (aqueous KOH solution, 0.5%, 2 h) produced kaempferol3-O-L-arabinopyranoside (juglanin) of formula C20H18O10, mp 224–226°C (EtOH). UV spectrum (MeOH, max, nm): 266, 350. Stepwise acid hydrolysis (15% AcOH, 2 h) of 2 produced the intermediate monoglycoside kaempferol-7-O-Lrhamnopyranoside (rhamnoisorobinin) of formula C21H20O10, mp 170–173°C (EtOH). Kaempferol-3-O-L-arabinopyranoside-7-O-L-rhamnopyranoside (2). 1Í NMR spectrum (600 MHz, DMSO-d6, ppm, J/Hz): 6.45 (1Í, d, J = 2.2, Í-6), 6.83 (1Í, d, J = 2.2, Í-8), 8.12 (1Í, d, J = 8.8, Í-2 , 6 ), 6.9 (1Í, d, J = 8.8, Í-3 , 5 ), 5.35 (1Í, d, J = 5.5, Í-1 ), 3.75 (1Í, dd, J = 6.9, 5.5, Í-2 ), 3.53 (1Í, dd, J = 6.9, 3.0, Í-3 ), 3.66 (1Í, m, Í-4 ), 3.58 (2Í, dd, J = 11.6, 5.5, Í-5 ), 3.22 (2Í, dd, J = 11.6, 2.2, Í-5 ), 5.56 (1Í, d, J = 1.7, Í-1 ), 3.85 (1Í, br.s, Í-2 ), 3.64 (1Í, dd, J = 9.4, 3.3, Í-3 ), 3.30 (1Í, br.t, J = 9.4, Í-4 ), 3.43 (1Í, dq, J = 9.4, 6.2, Í-5 ), 1.12 (3Í, d, J = 6.1, Í-6 ). 13C NMR spectrum (150 MHz, DMSO-d6, ppm): 160.2 (Ñ-2), 133.9 (Ñ-3), 177.7 (Ñ-4), 160.9 (Ñ-5), 99.4 (Ñ-6), 161.6 (Ñ-7), 94.6 (Ñ-8), 155.9 (Ñ-9), 105.6 (Ñ-10), 120.6 (Ñ-1 ), 131.1 (Ñ-2 , 6 ), 115.3 (Ñ-3 , 5 ), 156.8 (Ñ-4 ), 101.2 (Ñ-1 ), 70.8 (Ñ-2 ), 71.5 (Ñ-3 ), 66.0 (Ñ-4 ), 64.2 (Ñ-5 ), 98.4 (Ñ-1 ), 69.8 (Ñ-2 ), 70.1 (Ñ-3 ), 71.6 (Ñ-4 ), 69.8 (Ñ-5 ), 17.9 (Ñ-6 ) [7].

Flavonoids from Stachys annua Growing in Azerbaijan

The new acylated flavonoid bioside 4′-O-methylisoscutellarein-7-O-[4′′′-O-acetyl]allopyranosyl-(1→2)-glucopyranoside (1) was isolated from the aerial parts of Stachys annua L. (Lamiaceae). Subterranean organs yielded for the first time 4′-O-methylisoscutellarein (2) and 4′-O-methylisoscutellarein-7-O-[6′′-Oacetyl] allopyranosyl-(1→2)-glucopyranoside (3). Chemical structures of the isolated compounds were elucidated using NMR spectroscopy.

Biologically Active Compounds from Berberis libanotica

The search for new sources of biologically active compounds (BACs) prompted us to use LC/MS to determine their qualitative and quantitative contents in the aerial organs (branches and leaves) of the endemic species Berberis libanotica (Berberidaceae). Also, the antitumor activity of the various extracts was determined in vitro. Alkaloids of the genus Berberis L. possess antitumor, cytotoxic, anti-inflammatory, hepatoprotective, and hypotensive activity [1–3]. Tincture of B. libanotica aerial organs is used in folk medicine as a topical medicine for rheumatic diseases and muscle aches [4]. Raw material for the study was collected in September 2011 in the north of Lebanon in the Bsharri cedar zone at 1400–1700 m elevation. Powder of ground air-dried aerial organs (branches and leaves) of B. libanotica was extracted with H2O (folk method), EtOH (60%), and CH2Cl2 in the presence of NH4OH. Decoction was prepared by mixing H2O (150 mL) and powdered aerial organs (10.0 g) and boiling for 15 min. The filtered hot mixture was cooled and lyophilized to afford dry extract (1.12 g). Powdered aerial organs (10.0 g) were macerated with EtOH (60%, 150 mL) by mixing at room temperature and stirring for 24 h on a magnetic stirrer. The mixture was vacuum filtered. The EtOH was evaporated. The aqueous residue was lyophilized to afford dry extract (1.06 g). The CH2Cl2 extract was prepared by moistening of powdered aerial organs (10.0 g) with NH4OH solution (25%) for 2 h, adding CH2Cl2 (150 mL), macerating, and extracting for 24 h on a magnetic stirrer. The organic phase was separated and concentrated in vacuo at 40°C to afford dry extract (0.2 g). Each extract (20 L, 0.2 mg/mL for alkaloids and 0.4 mg/mL for phenolic acids) was analyzed by HPLC. The standards were the alkaloids hydroxyacanthine, berbamine, jatrorrhizine, palmatine, and berberine (0.05 mg/mL). The same final concentration was used for phenolic acids, chlorogenic, caffeic, and ferulic. Each standard solution (10 L) was analyzed. All analyzed samples were filtered through a 0.45 m nylon filter. Measurements were made at 235 nm for alkaloids and 220 nm for other compounds. The published HPLC method [5] was modified by replacing orthophosphoric acid by formic acid. The mobile phase consisted of H2O (A) and MeCN (B) acidified by formic acid (2%). The flow rate was 1.0 mL/min. Gradient elution (%B) used 0–2 min (15), 2–32 min (15–37), 32–35 min (37), 35–40 min (37–50), 40–42 min (50–15), 42–50 min (15). The relative qualitative contents of the main compounds of each extract were determined by HPLC-DADMS [5]; the quantitative contents, by calculating the ratio of peak areas of the compound and the corresponding standard. All experimental results were averages of three analyses (Table 1). Five alkaloids (hydroxyacanthine, berbamine, jatrorrhizine, palmatine, and berberine) that were described from other Berberis species and three phenolic acids (chlorogenic, caffeic, and ferulic) were identified for the first time in aerial organs of B. libanotica [6]. The activities of three extracts on the proliferation and colony formation of HT-29 human colon cancer cells were studied using the MTT assay. Only the CH2Cl2 extract showed significant activity [24.5 g/mL; control 5-fluorouracil (5-FU), 0.094 g/mL] that was related exclusively to the alkaloids and was dose-dependent.

Chemical Constituents from Roots of Cephalaria media

Twenty of the 60 species of Cephalaria L. distributed in southern Europe and in western and eastern regions of the Mediterranean and South Africa grow in the Caucasus, including 12 in Azerbaijan [1]. In continuation of phytochemical research on the family Dipsacaceae growing in the Republic of Azerbaijan, we studied roots of Cephalaria media Litv. The chemical composition of this endemic species growing in Azerbaijan has not previously been studied [2]. Dried ground roots of C. media (200.0 g) were extracted (2 ) with MeOH (1:10). The resulting extracts were filtered through cotton-wool and evaporated in a rotary evaporator. The dry residue was treated with H2O (100– 150 mL), thoroughly shaken, and poured into flasks (20 mL) for lyophilization. Dry extract (2 g) was obtained after 3 d and mixed with polyamide (10 g), treated with H2O (500 mL), placed onto a column, eluted first with H2O (500 mL) and then MeOH (10%, 500 mL; 50%, 500 mL; 100%, 300 mL). The process was repeated three times to afford 20 fractions that were combined if identical to give F4 + F5, F6 + F7 + F8, F9 + F10 + F11 + F12, F13 + F14 + F15 + F16, and F17 + F18 + F19 + F20. Fraction F4-5 was purified by flash chromatography by mixing it (1.0577 g) with silica gel (6 g) and placing the mixture on a silica gel column (40 g). Fractions (10 mL) with peak = 210 nm were collected over 30 min using CH2Cl2–MeOH–H2O (40:7:1) and then MeOH (100%) for 10 min at flow rate 20 mL/min. The fractions were monitored by TLC using CH2Cl2–MeOH–H2O (35:12:2) and combined in the order 1–5, 6–10, 11–12, 13, 14–17, 18–20, 19, 21–28, 29–30, 31–34, and 35–39. Fractions 29–30 (3 mg) afforded 1 that was identified as loganoside. Fractions 31–34 (15 mg) also contained loganoside with impurities. Fraction 19 (6.5 mg) gave 2 that was identified as sweroside [3].