Béatrice Baghdikian

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.

α‐hederin potentiates 5‐FU antitumor activity in human colon adenocarcinoma cells

The aim of this study was to investigate the ability of α‐hederin to improve the efficacy of widely prescribed 5‐fluorouracil (5‐FU) in a human colon adenocarcinoma model. Drug combinations of α‐hederin and 5‐FU using both fixed‐concentration and combination index methods were performed in vitro in HT‐29 cells. The results showed that α‐hederin at sub‐IC50 cytotoxic concentrations enhanced 5‐FU cytotoxicity about 3.3‐fold (p < 0.001). Simultaneous combination of α‐hederin and 5‐FU at their IC50 ratio showed either a synergistic effect at a moderate cytotoxic range (25% of cell growth inhibition) or an antagonistic effect at a high level of growth inhibition. The data indicate therefore that it is possible to optimize colorectal cancer cell sensitivity to 5‐FU with α‐hederin. Copyright

Ethnobotany, phytochemistry and pharmacology of Stephania rotunda Lour.

ETHNOPHARMACOLOGICAL RELEVANCE Stephania rotunda Lour. (Menispermaceae) is an important traditional medicinal plant that is grown in Southeast Asia. The stems, leaves, and tubers have been used in the Cambodian, Lao, Indian and Vietnamese folk medicine systems for years to treat a wide range of ailments, including asthma, headache, fever, and diarrhoea. AIM OF THE REVIEW To provide an up-to-date, comprehensive overview and analysis of the ethnobotany, phytochemistry, and pharmacology of Stephania rotunda for its potential benefits in human health, as well as to assess the scientific evidence of traditional use and provide a basis for future research directions. MATERIAL AND METHODS Peer-reviewed articles on Stephania rotunda were acquired via an electronic search of the major scientific databases (Pubmed, Google Scholar, and ScienceDirect). Data were collected from scientific journals, theses, and books. RESULTS The traditional uses of Stephania rotunda were recorded in countries throughout Southeast Asia (Cambodia, Vietnam, Laos, and India). Different parts of Stephania rotunda were used in traditional medicine to treat about twenty health disorders. Phytochemical analyses identified forty alkaloids. The roots primarily contain l-tetrahydropalmatine (l-THP), whereas the tubers contain cepharanthine and xylopinine. Furthermore, the chemical composition differs from one region to another and according to the harvest period. The alkaloids exhibited approximately ten different pharmacological activities. The main pharmacological activities of Stephania rotunda alkaloids are antiplasmodial, anticancer, and immunomodulatory effects. Sinomenine, cepharanthine, and l-stepholidine are the most promising components and have been tested in humans. The pharmacokinetic parameters have been studied for seven compounds, including the three most promising compounds. The toxicity has been evaluated for liriodenine, roemerine, cycleanine, l-tetrahydropalmatine, and oxostephanine. CONCLUSION Stephania rotunda is traditionally used for the treatment of a wide range of ailments. Pharmacological investigations have validated different uses of Stephania rotunda in folk medicine. The present review highlights the three most promising compounds of Stephania rotunda, which could constitute potential leads in various medicinal fields, including malaria and cancer.

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.

New sesquiterpene acid and inositol derivatives from Inula montana L.

A phytochemical investigation of the ethanol extract of leaves and flowers of Inula montana L. led to the isolation of one new sesquiterpene acid called Eldarin (1) and four new inositol derivatives, Myoinositol,1,5-diangelate-4,6-diacetate (2), Myoinositol,1,6-diangelate-4,5-diacetate (3), Myoinositol-1-angelate-4,5-diacetate-6-(2-methylbutirate) (4), Myoinositol-1-angelate-4,5-diacetate-6-isovalerate (5) isolated for the first time, along with eleven known compounds described for the first time in Inula montana, 1β-Hydroxyarbusculin A (6), Artemorin (7), Santamarin (8), Chrysosplenol C (9), 6-Hydroxykaempferol 3,7-dimethyl ether (10), Reynosin (11), Calenduladiol-3-palmitate (12), Costunolide (13), 4-Hydroxy-3,5-dimethoxybenzenemethanol (14), 9β-Hydroxycostunolide (15) and Hispidulin (16). Structural elucidation has been carried out by spectral methods, such as 1D and 2D NMR, IR, UV and HR-ESI-MS. These compounds have been tested in vitro for anti-inflammatory and cytotoxic activity on macrophages RAW 264.7. As a result, compounds 2, 3, 7, 13, 14, 15 and 16 showed a release of NO with IC50 value <30μM on macrophages.

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 Chamaenerion angustifolium and Stachys annua Growing in Azerbaidzhan

A search for possible new sources of biologically active compounds led to the aerial parts of Chamaenerion angustifolium L. Holub (Onagraceae Juss.) and Stachys annua L. (Lamiaceae) [1, 2]. Many representatives of the genera Chamerion (Rafin.) Rafin. and Stachys L. are used in folk medicine in various countries [3, 4]. Raw material of C. angustifolium was collected at the end of July 2013 between Shamakhi and Ismailli Districts; S. annua, at the end of May 2012 in the vicinity of Pirqulu village, Shamakhi District, Republic of Azerbaidzhan. Ground air-dried aerial parts of C. angustifolium (1.5 kg) were extracted with EtOH (80%). The extracts were combined and evaporated to an aqueous residue that was worked up successively with CHCl3 and EtOAc. Chromatography of the CHCl3 extract over a column of Al2O3 with elution by hexane–CHCl3 isolated compounds 1 and 2. Recrystallization of the EtOAc extract from EtOH afforded compound 3. The filtrate was evaporated to dryness. The residue was hydrolyzed by H2SO4 (3%, 4 h). The hydrolysate was diluted with ice water and filtered. The filtrate was dried and chromatographed over a column with polyamide sorbent with elution by CHCl3–MeOH with increasing volume of the latter to afford two flavonoid aglycons 4 and 5. -Sitosterol (1), C29H50O, mp 140–142°C (EtOH), [ ]D 20 –40° (c 0.6, CHCl3), mp of acetate 128–130°C (aq. EtOH) [5]. Ursolic acid (2), C30H48O3, mp 280–282°C (EtOH), [ ]D 20 +56° (c 0.6, MeOH). The IR spectrum showed absorption bands at 3300–3200 (OH) and 1705 cm–1 (carboxylic acid) [5]. Ellagic acid (3), C14H6O8, mp 350°C (dec.), Rf 0.44 and 0.1 using BAW (40:12.5:29) and AcOH (20%), respectively. 1Í NMR spectrum (600 MHz, DMSO-d6, ppm, J/Hz): 7.45 (1Í, s, Í-5, 5 ). 13C NMR spectrum (150 MHz, DMSO-d6, ppm): 112.4 (Ñ-1, 1 ), 136.4 (Ñ-2, 2 ), 139.7 (Ñ-3, 3 ), 148.2 (Ñ-4, 4 ), 110.3 (Ñ-5, 5 ), 107.6 (Ñ-6, 6 ), 159.2 (Ñ-7, 7 ) [6, 7]. Kaempferol (3,5,7,4 -tetrahydroxyflavone) (4). C15H16O6, mp 274–276°C (EtOH), mp of tetraacetate 182–189°C (MeOH–CHCl3) [6, 7]. Quercetin (3,5,7,3 ,5 -pentahydroxyflavone) (5). C15H10O7, mp >300°C (EtOH), mp of pentaacetate 198–200°C [6, 7]. Compounds 6–8 were isolated from air-dried and ground aerial parts (0.9 kg) of S. annua by extraction with EtOH (80%). Compound 6 was identified as -sitosterol; 7, ursolic acid [5]. 4 -O-Methylisoscutellarein-7-O-[6 -O-acetyl]-D-allopyranosyl(1 2 )-D-glucopyranoside (8). C30H34O17, mp 262–264°C (EtOH), [ ]D 20 –96° (EtOH), lemon-yellow crystals, soluble in aq. EtOH, DMF, and Py; poorly soluble in EtOH and H2O; insoluble in CHCl3 and EtOAc. 1Í NMR spectrum (600 MHz, DMSO-d6, ppm, J/Hz): 6.91 (1Í, s, Í-3), 6.70 (1Í, s, Í-6), 8.09 (2Í, d, J = 8.8, Í-2 , 6 ), 7.13 (2Í, d, J = 8.8, Í-3 , 5 ), 3.87 (3H, s, ÎÑÍ3), 5.08 (1Í, d, J = 7.7, Í-1 ), 3.58 (1Í, br.t, J = 7.7, Í-2 ), 3.51 (1Í, br.t, J = 8.8, Í-3 ), 3.25 (1Í, m, Í-4 ), 3.47 (1Í, m, Í-5 ), 3.74 (1Í, br.d, J = 11.0, Í-6 ), 3.49 (1Í, m, Í-6 ), 4.92 (1Í, d, J = 7.7, Í-1 ), 3.25 (1Í, m, Í-2 ), 3.91 (1Í, t, J = 2.8, Í-3 ), 3.41 (1Í, dd, J = 9.8, 2.8, Í-4 ), 3.88 (1Í, m, Í-5 ), 4.05 (1Í, dd, J = 12.1, 2.2, Í-6 ), 4.01 (1Í, dd, J = 12.1, 5.0, Í-6 ), 1.87 (s, O-Àc). 13C NMR spectrum (150 MHz, DMSO-d6, ppm): 163.7 (Ñ-2), 103.4 (Ñ-3), 182.5 (Ñ-4), 152.2

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.