Field evaluation of in vitro-induced tetraploid and diploid Centella asiatica (L.) Urban

Centella asiatica—a medicinal plant that produces high-value active triterpenoids—is in increasing demand by the pharmaceutical and cosmetic industries. The aim of this study was to field-test one induced tetraploid and three diploid C. asiatica lines for the selection of high-quality plants with high phytomass and triterpenoid content and to determine their optimal harvesting time. All tested C. asiatica were micropropagated using an established protocol. One-month-old plantlets were acclimatized for the field experiment. The plants were grown in a randomized complete block design (RCBD) with three replications, ten plantlets per replication, and the experimental bed site was 0.6 × 1.0 m. Growth parameters, phytomass and the amounts of four active triterpenoids were evaluated. All lines exhibited the highest growth, yields and triterpenoids at 4 months after cultivation. The tetraploid line showed significantly better characteristics, i.e., larger leaf area, leaf width, petiole length, and greater yields, than diploid lines. Dry weight per cultivated area (77.53 ± 3.07 g/m²) and total triterpenoids (15.38 ± 0.76 % dry weight) were increased significantly in tetraploid plants of C. asiatica. Furthermore, the harvesting time had an effect on the yield and triterpenoid content (P < 0.001). In all tetraploid and diploid lines, the yields and triterpenoid content per cultivated area reached their maximum at 4 months after planting. Our results demonstrated that polyploidy induction is a beneficial tool that can be used to improve the medicinal value of C. asiatica.     

积雪草苷类化合物对神经系统药理作用研究进展

积雪草总苷是积雪草的主要活性成分,其中的单体成分包括积雪草苷、羟基积雪草苷和积雪草酸等,在临床上主要用于治疗皮肤损伤,促进创面愈合以及瘢痕修复等。近年来发现积雪草苷类化学成分对神经系统也存在着多种药理活性,如抗抑郁、抗痴呆、对脊髓损伤以及脊髓神经细胞变性的保护、对抗脑缺血损伤以及镇痛、抗惊厥等。综述积雪草苷类化学成分对神经系统的药理作用研究进展,希望为该类化合物治疗神经系统疾病的研究与应用及新药开发提供理论依据。     

Changes in the contents of main secondary metabolites in two Turkish Hypericum species during plant development

Context: The genus Hypericum (Guttiferae) has received considerable scientific interest as a source of biologically active compounds.

Objective: The study determined the morphogenetic and ontogenetic variation in the main bioactive compounds of two Hypericum species, namely, Hypericum aviculariifolium subsp. depilatum var. depilatum (Freyn and Bornm.) Robson var. depilatum and Hypericum orientale L. through HPLC analyses of whole plants as well as individual plant parts (stems, leaves, and reproductive tissues).

Materials and methods: The plant materials were harvested at five phenological stages: vegetative, floral budding, full flowering, fresh fruiting, and mature fruiting; dried at room temperature, then assayed for chemical content.

Results: In H. aviculariifolium, no kaempferol accumulation was observed and the highest level of hypericin, pseudohypericin, and quercitrin was reached at full flowering (0.71, 1.78, and 4.15?mg/g DW, respectively). Plants, harvested at floral budding produced the highest amount of rutin, hyperoside, and isoquercitrine (32.96, 2.42, 1.52?mg/g DW, respectively). H. orientale did not produce hypericin, pseudohypericin, or kaempferol. Rutin, hyperoside, and isoquercetine levels were the highest at floral development (1.76, 11.85, and 1.21?mg/g DW, respectively) and plants harvested at fresh fruiting produced the highest amount of quercitrine and quercetine (0.20 and 1.30?mg/g DW, respectively).

Discussion: For the first time, the chemical composition of the Turkish species of Hypericum was monitored during the course of ontogenesis to determine the ontogenetic and morphogenetic changes in chemical content.

Conclusions: Plant material should be harvested during flower ontogenesis for medicinal purposes in which the content of many bioactive substances tested reached their highest level.     

Matrix metalloproteinase,hyaluronidase and elastase inhibitory potential of standardized extract of Centella asiatica

Abstract

Context: Centella asiatica (L.) Urban (Apiaceae), a valuable herb described in Ayurveda, is used in the indigenous system of medicine as a tonic to treat skin diseases.

Objective: Centella asiatica methanol extract and its ethyl acetate, n-butanol and aqueous fraction, were subjected for the evaluation of skin care potential through the in vitro hyaluronidase, elastase and matrix metalloproteinase-1 (MMP-1) inhibitory assay.

Materials and methods: The C. asiatica plant was extracted with methanol and fractionated with ethyl acetate, n-butanol and water. The enzymatic activities were evaluated using ursolic acid and oleanolic acid as standards. Isolate molecule asiaticoside was quantified in the crude extract and fractions through high-performance liquid chromatography (HPLC) and structural was characterized by liquid chromatography–mass spectroscopy (LC–MS) and ¹H nuclear magnetic resonance (NMR). Isolated compound was also evaluated for in vitro enzyme assays.

Results: Extract exhibited anti-hyaluronidase and anti-elastase activity with IC₅₀ of 19.27?±?0.37 and 14.54?±?0.39?µg/mL, respectively, as compared to ursolic acid. Centella asiatica n-butanol fraction (CAnB) and isolated compound showed significant hyaluronidase (IC₅₀?=?27.00?±?0.43 and 18.63?±?0.33?µg/mL) and elastase (IC₅₀?=?29.15?±?0.31 and 19.45?±?0.25?µg/mL) inhibitory activities, respectively, and also showed significant MMP-1 inhibition (p?<?0.05 and p?<?0.01).

Discussion and conclusion: n-Butanol fraction was found to be most effective among the all fractions from which asiaticoside was isolated and further quantified by HPLC. This work concludes that the asiaticoside from C. asiatica may be a prospective agent for skin care.     

Inhibitory effect of aqueous extract of different parts of unripe pawpaw (Carica papaya) fruit on Fe2+-induced oxidative stress in rat pancreas in vitro

Abstract

Context: Carica papaya L. (Caricaceae) is widespread throughout tropical Africa; it is cultivated for its fruits and it is eaten in various ways.

Objective: This study sought to investigate the inhibitory effect of the aqueous extract of different parts of unripe pawpaw fruit on Fe²⁺-induced lipid peroxidation in rat’s pancreas in vitro.

Materials and methods: The aqueous extract of the unripe pawpaw fruit parts; peel (PG), seed (SG), flesh (FG), flesh with peel (FPG) and a combination of equal amount of all parts (CG) were prepared, the total phenolic content and the antioxidant activities of the extracts were then evaluated using various spectrophotometric methods.

Result: PG had the highest total phenol content (1.24?mg GAE/g), flavonoid content (0.63?mg QUE/g), reducing power (7.07?mg AAE/g) and Fe²⁺ chelating ability while the SG had the highest 1,1-diphenyl-2-picrylhydrazyl radical scavenging ability. Furthermore, all the extracts caused a significant decrease (p?<?0.05) in the malondialdehyde contents in the pancreas with SG (IC₅₀?=?4.25?mg/mL) having the highest inhibitory effect on Fe²⁺-induced lipid peroxidation.

Discussion and conclusion: This protective effect of the extracts on Fe²⁺-induced lipid peroxidation in rat pancreas could be attributed to their phenolic compounds and, the possible mechanism may be through their antioxidant activities. However, the effect of combination of different parts of unripe pawpaw fruit in equal amount (w/w) on the inhibition of Fe²-induced lipid peroxidation in rat pancreas exhibited additive properties.     

Seasonal variation in the content of a febrifugine and isofebrifugine alkaloid mixture in aerial parts of <Emphasis Type="Italic">Hydrangea macrophylla</Emphasis> var. <Emphasis Type="Italic">Otaksa</Emphasis>, with special reference to its antimalarial activity

Febrifugine and isofebrifugine alkaloid mixtures extracted from the leaves and buds of Hydrangea macrophylla var. Otaksa, collected during different months, in Japan, were quantified using high-performance liquid chromatography. Leaves collected during the flowering season, namely from June to August, contained 0.16–0.31 mg/g of the alkaloid mixture, whereas those collected from September to December had less than 0.03 mg/g of the mixture. However, extracts of buds harvested from October to February contained a consistently larger amount (more than 0.49 mg/g) of the alkaloids. Hot-water extracts from the leaves and buds collected during different seasons were evaluated for antimalarial activity against Plasmodium yoelii 17XL in mice. The extract of leaves collected in August demonstrated high antimalarial activity, and all mice that received the extract survived the infection. In contrast, the extract of leaves collected in December showed little activity. The extract of buds collected in December cleared parasites, but with subsequent mortality to mouse. The present results show that the amount of antimalarial agent—febrifugine and isofebrifugine mixture—in H. macrophylla var. Otaksa is both part- and season-dependent, suggesting that the choice of plant parts and their harvesting season are important factors worth considering in the pharmacological use of medicinal plants.     

Effects of oleanolic acid and maslinic acid on hyperlipidemia

In the present study, we examined the therapeutic effects of pentacyclic triterpenes, oleanolic acid (OA) and maslinic acid (MA), on hyperlipidemia in a high‐cholesterol diet model. Hyperlipidemia was induced in male Sprague‐Dawley rats by feeding with a high‐cholesterol diet (HCD) for 30 days. MA and OA were supplemented (100 mg/kg body wt/day) during the last 15 days. The levels of serum total cholesterol (TC), triglyceride (TG), high‐density lipoprotein‐cholesterol (HDL‐C), and low‐density lipoprotein‐cholesterol (LDL‐C) increased in hyperlipidemic rats. An apparent increase in the expression of Acyl‐CoA cholesterol acyltransferase (ACAT) mRNA was seen in HCD‐fed rats. The activities of hepatic marker enzymes that serve as indices of cellular injury were also altered in HCD‐fed rats. Treatment with triterpenes modulated the abnormalities induced by hyperlipidimia. Lipid accumulation was decreased in histological findings. Elevated hepatic glycogen content (P<0.05) in triterpene groups was observed compared with HCD‐fed groups. Furthermore, ACAT gene expression was suppressed compared with hyperlipidemia‐induced groups without triterpenes. It can be concluded that triterpene treatment possesses therapeutic effects on diet‐induced hyperlipidemia by inhibiting the intestinal absorption and storage of cholesterol. Drug Dev Res 68:261–266, 2007. © 2007 Wiley‐Liss, Inc.     

Phenolic composition,antioxidant and anti-acetylcholinesterase activities of the Tunisian Scabiosa arenaria

Abstract

Context: There is a need for the discovery of novel natural antioxidants and acetylcholinesterase inhibitors (AChEIs) that are safe and effective at a global level. This is the first study on antioxidant and anti-acethylcholinesterase activity of Scabiosa arenaria Forssk (Dipsacaceae).

Objective: The antioxidant potential and anti-acetylcholinesterase (AChE) activity of S. arenaria were investigated.

Material and methods: The crude, ethyl acetate (EtOAc), butanol (n-BuOH) and water extracts prepared from flowers, fruits and stems and leaves of S. arenaria were tested to determine their total polyphenol content (TPC), total flavonoid content (TFC), total condensed tannin content (CTC) and their antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), reducing power and β-carotene bleaching inhibition activity. Anti-AChE activity was also determined.

Results: EtOAc and n-BuOH fractions of fruits had both the highest (TPC) (269.09?mg gallic acid equivalents/g dry weight). The crude extract of stems and leaves had the highest TFC (10.9?mg quercetin equivalent/g dry weight). The n-BuOH fraction of stems and leaves had the highest CTC (489.75?mg catechin equivalents/g dry weight). The EtOAc fraction of flowers exhibit a higher activity in each antioxidant system with a special attention for DPPH assay (IC₅₀?=?0.017?mg/mL) and reducing power (EC₅₀?=?0.02?mg/mL). The EtOAc and n-BuOH fractions of stems and leaves showed strong inhibition of AChE (IC₅₀?=?0.016 and 0.029?mg/mL, respectively).

Discussion and conclusions: These results suggest the potential of S. arenaria as a possible source of novel compounds and as an alternative antioxidant and AChEIs.     

Microbial transformation of asiatic acid by Alternaria longipes

Asiatic acid is a major pentacyclic triterpene isolated from Centella asiatica. It shows a variety of bioactivities. In order to obtain its derivatives, potentially useful for detailed pharmacological studies, the substrate was subjected to incubations with selected micro-organisms. In this work, asiatic acid was converted into three new compounds: 2α,3β,23,30-tetrahydroxyurs-12-ene-28-oic acid (1), 2α,3β,22β,23-tetrahydroxyurs-12-ene-28-oic acid (2), and 2α,3β,22β,23,30-pentahydroxyurs-12-ene-28-oic acid (3) by the fungus Alternaria longipes AS 3.2875. The structures of the three metabolites were determined by 1D and 2D NMR spectral data.     

Anticancer activities of the extract from Longkong (Lansium domesticum) young fruits

Context: “Longkong”(Lansium domesticum Corr., Family: Meliaceae) is a fruit found in the south of Thailand. This plant has been used in traditional medicines.

Objectives: To investigate the antiproliferative activities and the phytoconstituents of Longkong extracts.

Materials and methods: Cytotoxicity and apoptotic activity of 48 extracts were tested using the SRB assay and acridine orange (AO)/ethidium bromide (EB) staining, respectively. The extracts which gave the highest anticancer activity were selected to prepare the semipurified extracts and analysis for the constituents by gas chromatography–mass spectrometry (GC/MS).

Results: The highest percentage yield (59.38%) was from the cold water extract of Longkong ripe fruits (RFWC). The highest total phenolic and flavonoid contents were observed in cold and hot methanol extract of Longkong stalks (STMC and STMH). The hot and cold chloroform young Longkong fruit extracts (YFCH and YFCC) exhibited a cytotoxic effect (IC₅₀ < 1 mg/mL) against cancer cells. For apoptotic induction, YFCH demonstrated the highest activity against KB of 13.84 ± 4.21% at 0.5 mg/mL which was 0.88 and 1.35 times of cisplatin and 5-FU, respectively, while apoptotic cells in HT-29 were 8.68 ± 1.85% at 5 mg/mL, which was 0.61 and 1.43 times of cisplatin and 5-FU, respectively. YFCC showed the highest apoptotic effect against KB cells at 10.70 ± 2.15% at 0.5 mg/mL, which was 0.68 and 1.07 times of cisplatin and 5-FU, respectively. The major phytoconstituents in YFCH were hexadecanoic acid (11.53%) and ethyl oleate (10.58%).

Discussion and conclusion: The crude extracts of Longkong showed anticancer activities and may provide new lead compounds for the development of anticancer products.     

Antimicrobial bioassay-guided fractionation of a methanol extract of Eupatorium triplinerve

Abstract

Context: Eupatorium triplinerve Vahl (Asteraceae), popularly known as Japana, is widely used in folk medicine, due its analgesic, anticoagulant, antianorexic, antiparasitic, anthelmintic, sedative, antifungal, and antibacterial properties.

Objective: The present study evaluated the chemical composition and antimicrobial activity of E. triplinerve extracts from different parts of the plant and identified the extract with the highest antimicrobial potential.

Materials and methods: Extracts were obtained by maceration of all parts of plant, and subjected to bioassay-guided fractionation of methanol extract by partition column chromatography. The major chemical groups, saponins, reducing sugars, alkaloids, steroids, triterpenoids, phenols, tannins, flavonoids, and others were screened by standard techniques. The antimicrobial activity of the different extracts was performed by microdilution assay and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were reported.

Results: Phytochemical screening of hydroalcoholic extract from all parts of E. triplinerve identified mainly steroids, coumarins, alkaloids, saponins, tannins, depsides and absence of polysaccharides and flavonoids. The methanol extract of leaves presented the highest content of coumarins and lower MIC values of 62 and 75?µg/mL against Enterococcus faecalis and Staphylococcus aureus, respectively. In addition, its non-polar fractions showed antimicrobial activity with MIC ranging from 16 to 125?µg/mL against Gram-negative bacteria, mainly Escherichia coli.

Discussion and conclusion: Data showed that non-polar fractions of E. triplinerve methanolic extract has better antimicrobial activity and most likely depends on the presence of several compounds, such as depsidones, coumarins, saponins, and triterpenes on crude extract. The results can be exploited largely in research of new antibacterial agents.     

不同采收期柴胡中挥发油及总皂苷的含量测定

目的:测定不同采收期柴胡中挥发油及总皂苷的含量。方法:采用挥发油提取器提取柴胡中的挥发油;以柴胡皂苷a为指标,采用可见分光光度法分别对不同采收期柴胡中的总皂苷进行含量测定。结果:柴胡挥发油的含量以7月份最高,11月份最低;柴胡总皂苷的含量以5月份最高,9月份最低。结论:本方法简便、快速,可以为柴胡确定最佳采收期提供科学依据。     

Evaluation of seasonal changes of triterpenic acid contents in Viscum album from different host trees

Context: Viscum album L. (Loranthaceae) is a semi-parasitic plant used in pharmacy and medicine mostly for its hypotensive and anticancer activity. The effects may be related to the presence of triterpenic acids, such as betulinic (BA) and oleanolic (OA) acids.

Objectives: In our investigations the content of triterpenic acids in V. album from different host trees depending on the season of harvest was determined.

Material and methods: V. album herb was dried and extracted with ethyl acetate using ultrasound energy. The reversed phase HPLC-PDA method was used for the analysis of triterpenic acids. The structure of the target components was confirmed by mass spectrometry with an electrospray ionization source.

Results: Diversity in the content of both compounds was noted; however, OA was the dominant triterpenic acid and the amount thereof was ～10 times higher than that of BA. The analysis of changes in the amount of triterpenic acids during the spring-winter period revealed the highest content of OA in summer (from 6.84 to 13.65?mg/g). In turn, in the other seasons of harvest, the content was in the range of 4.41–9.83, 6.41–9.56 and 5.59–12.16?mg/g for spring, autumn and winter, respectively. In most cases, a similar tendency was observed for BA.

Discussion and conclusion: In most cases, the highest amount of the investigated compounds was found in summer; thus, this period seems to be optimal for acquisition of plant material rich in triterpenic acids.     

Changes in bioactive components related to the harvest time from the spicas of Prunella vulgaris

Context: Prunella vulgaris L. (Labiatae) is a perennial plant common in China and Europe and is rich in rosmarinic acid (RA), ursolic acid (UA), and oleanolic acid (OA). The dried spica of P. vulgaris has been used as traditional medicine in China for over a hundred years. To our best knowledge, no study has been conducted to determine the influence of harvesting time on concentrations of bioactive compounds of P. vulgaris.

Objective: In the current study, changes in the bioactive compounds present in spicas were investigated at five harvest times over 2 months.

Materials and methods: Plant material were collected at five fixed dates: 5th May, 20th May, 7th June, 15th June, and 25th June and assayed for chemical contents by high-performance liquid chromatography (HPLC).

Results: Among the different harvest times, the highest levels of RA (56.81 mg·g^?1), UA (2.77 mg·g^?1), and OA (0.91 mg·g^?1) were found on 5th May, whereas the lowest levels of RA (1.66 mg·g^?1), UA (2.27 mg·g^?1), and OA (0.43 mg·g^?1) were observed on 25th June.

Discussion and conclusion: As each medicinal product has its own content requirement for different bioactive components, the optimum harvest time might be determined according to the accumulation dynamics of target compound in dried spicas of P. vulgaris. These results may be useful for determining the optimal harvest time when bioactive components are at the maximum level, which is in early May.     

Three new lupane-type triterpenes from Ceriops tagal

Three new lupane-type triterpenes, 3α-O-trans-feruloylbetulinic acid (1), 3α-O-trans-coumaroylbetulinic acid (2) and 3β-O-cis-feruloylbetulin (3), together with 10 known triterpenes (4–13), were isolated from the aerial parts of the mangrove plant Ceriops tagal. The structures of the three new compounds were established by means of spectroscopic data analyses and chemical methods.     

Anti-proliferative and matrix metalloproteinase-2 inhibition of Longkong (Lansium domesticum) extracts on human mouth epidermal carcinoma

Abstract

Context: Longkong [Lansium domesticum Corr. (Meliaceae)] is a popular tropical plant producing economic edible fruits found mainly in Southeast Asia. However, limited information is available concerning anticancer activity of Longkong.

Objective: To investigate anticancer activities in human mouth epidermal carcinoma (KB) of Longkong extracts.

Materials and methods: Various parts of Longkong which was collected from Northern and Eastern of Thailand were extracted by the hot and cold processes using water, chloroform, and methanol. The extracts were tested for anti-oxidative activities and anti-proliferation as well as matrix metalloproteinase inhibition on KB cells.

Results: The hot water extract of seeds from Northern region (NSEWH), the cold water extract of old leaves from Northern region (NOLWC), and the hot chloroform extract of young leaves from Eastern region (EYLCH) showed the highest free radical scavenging, metal ion chelating, and lipid peroxidation inhibition with SC₅₀, MC₅₀ and IPC₅₀ values of 0.34?±?0.03, 0.47?±?1.60 and 0.86?±?0.31?mg/ml, respectively. The hot and cold chloroform extract of young fruits from Northern region (NYFCH and NYFCC) exhibited anti-proliferation effect against KB cells with IC₅₀ values of 603.45?±?55.35 and 765.06?±?46.19?mg/ml, respectively. NYFCC exhibited the highest pro- and active MMP-2 inhibition at 53.03?±?2.65 and 31.30?±?0.43%, more than all tested standard anticancer drugs except cisplatin.

Discussion and conclusion: The cold chloroform extract of young fruits from Northern region appeared to contain anticancer active compounds against KB cells because of its high anti-proliferation and MMP-2 inhibition activities.     

Effects of vegetative and flowering stages on the biosynthesis of artemisinin in <Emphasis Type="Italic">Artemisia</Emphasis> species

Artemisinin is an endoperoxide sesquiterpene lactone, and has been proven to be very effective in treating drug resistant cases of malaria, cancer, etc. The compound is obtained from Artemisia species. In the current study, the effects of vegetative and flowering stages on artemisinin production were studied, to determine the proper harvesting time of naturally growing Artemisia species with the highest levels of artemisinin. Eight Artemisia species along with two varieties were selected for this analytical work. The results showed that artemisinin content was high in the leaves of Artemisia indica, A. sieversiana, A. roxburghiana var. roxburghiana, A. roxburghiana var. gratae, and A. parviflora at the flowering stage. The highest artemisinin content was measured in the leaves of A. dracunculus var. dracunculus. Upon comparisons of artemisinin content among the individual plant species, the highest amount of artemisinin was again in A. dracunculus var. dracunculus followed by A. sieversiana when harvested at the flowering stage. In overall comparisons, the plants at the flowering stage showed high levels of artemisinin, which is deemed the optimum harvesting time of Artemisia species in Pakistan for maximum artemisinin content.     

Enhancement of anti-bacterial and anti-tumor activities of pentacyclic triterpenes by introducing exocyclic α,β-unsaturated ketone moiety in ring A

Ursolic acid, oleanolic acid, glycyrrhetinic acid, and betulinic acid, the representatives of pentacyclic triterpenes, were modified by introducing 2-methylene-3-oxo group as exocyclic α,β-unsaturated ketone moiety in ring A. The anti-bacterial and anti-tumor activities of these derivatives were assayed by comparing with the parent compounds. Results indicated that pentacyclic triterpenes carrying 2-methylene-3-oxo group in the ring A exhibited a significant improvement in anti-bacterial activity that was limited to Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. The four derivatives also showed an increased cytotoxicity against leukemia, lung, and breast cancer cell lines in a dose-dependent manner in vitro. U2 and O2 compounds showed strong apoptotic activities to lung carcinoma cell lines. The results for the first time provided scientific evidence for improvement of anti-bacterial and anti-tumor activities of pentacyclic triterpenes using derivatives of these compounds.     

Chemical composition and biological activities of Helicteres vegae and Heliopsis sinaloensis

Context: Helicteres vegae Cristóbal (Sterculiaceae) (Hv) and Heliopsis sinaloensis B.L. Turner (Asteraceae) (Hs) are endangered and poorly studied plant species; related plants have been used against chronic-degenerative and infectious diseases. Therefore, Hv and Hs could be sources of bioactive compounds against these illnesses.

Objective: To determine the chemical composition and biological activities (antioxidant, antimutagenic and antimicrobial) of Hv and Hs leaves (L) and stems (S).

Materials and methods: Methanol extracts (ME) of each plant/tissue were evaluated for their phytochemicals; phenolics (HPLC-DAD-ESI-MS); antioxidant activity (AA) (0.125–4?mg/mL) (DPPH, ABTS, ORAC and β-carotene discoloration); antimutagenicity (0.5 and 1?mg/plate) (Ames assay, tester strain Salmonella enterica serovar Typhimurium YG1024, 1-nitropyrene as mutagen); activity against human pathogens (1?mg/mL); and toxicity (0.01–2?mg/mL) (Artemia salina assay).

Results: All ME showed flavonoids and triterpenes/steroids. The ME-SHv had the highest content of total phenolics (TP) (2245.82?±?21.45?mg GAE/100?g d.w.) and condensed tannins (603.71?±?1.115?mg CE/100?g d.w.). The compounds identified were flavonoids (kaempferol 7-O-coumaroylhexoside, and two kaempferol 7-O-rhamnosylhexosides) and phenolics [rosmarinic acid, and 3′-O-(8″-Z-caffeoyl) rosmarinic acid]. The ME-LHs showed the highest content of flavonoids (357.88?mg RE/g d.w.) and phenolic acids (238.58?mg CAE/g d.w.) by HPLC. The ME-SHv showed the highest AA. All ME were strong antimutagens (63.3-85.7%). Only the Hs extracts were toxic (ME-LHs, LC₅₀?=?94.9?±?1.7?μg/mL; ME-SHs, LC₅₀?=?89.03?±?4.42?μg/mL).

Discussion and conclusions: Both Hv and Hs are potential sources of preventive and therapeutic agents against chronic-degenerative diseases.     

Production of eurycomanone from cell suspension culture of Eurycoma longifolia

Context: Eurycomanone is found in the Eurycoma longifolia Jack (Simaroubaceae) tree, exhibits significant antimalarial activity, improves spermatogenesis, suppresses expression of lung cancer cell tumour markers and regulates signalling pathways involved in proliferation, cell death and inflammation.

Objectives: Establishment of cell suspension culture of E. longifolia to determine the eurycomanone accumulation during cultures.

Materials and methods: Callus of E. longifolia was cultured in MS medium supplemented with 0.8% agar, 30/L sucrose, 1.25?mg/L NAA and 1?mg/L KIN for biomass production. Cell suspension culture was established by transferring friable calli to the same medium without agar. Eurycomanone content during cell culture was determined by HPLC with a C18 column, flow rate of 0.8?mL/min, run time of 17.5?min, detector wavelength of 254?nm. The stationary phase was silica gel and the mobile phase was acetonitric:H₂O. Roots of 5 year-old trees were used as the control.

Results: The cells from 3?g of inoculum increased in biomass with a maximum value of 16?g fresh weight (0.7?g dry weight) at 14th day of culture. The cell growth then decreased from day 14 to day 20. Eurycomanone was produced during culture from the beginning to 20th day, its highest content (1.7?mg/g dry weight) also obtained at 14th day (the control is 2.1?mg/g dry weight).

Discussion and conclusions: Cell suspension culture of E. longifolia is a suitable procedure to produce eurycomanone. The yield of eurycomanone biosynthesis in 14 days-old cells are relatively high, approximately 0.8 times the control.