Sequence, Structure, and Binding Site Analysis of Kirkiin in Comparison with Ricin and Other Type 2 RIPs.

Kirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly in cancer therapy. The complete amino acid sequence and 3D structure prediction of kirkiin are here reported. Gene sequence analysis revealed that kirkiin is encoded by a 1572 bp open reading frame, corresponding to 524 amino acid residues, without introns. The amino acid sequence analysis showed a high degree of identity with other Adenia RIPs. The 3D structure of kirkiin preserves the overall folding of type 2 RIPs. The key amino acids of the active site, described for ricin and other RIPs, are also conserved in the kirkiin A chain. Sugar affinity studies and docking experiments revealed that both the 1α and 2γ sites of the kirkiin B chain exhibit binding activity toward lactose and D-galactose, being lower than ricin. The replacement of His246 in the kirkiin 2γ site instead of Tyr248 in ricin causes a different structure arrangement that could explain the lower sugar affinity of kirkiin with respect to ricin.

A Polylactosamine Specific Lectin from Adenia hondala Induces Apoptosis and Necrosis in Human Epithelial Colon Cancer HT-29 Cells.

BACKGROUND: Altered expression of N-glycans such as polylactosamine is observed in colon cancer. AHL, a polylactosamine specific lectin from Adenia hondala from a medicinal plant from the Passifloraceae family has been reported earlier. OBJECTIVE: The aim of the present study is to study the interaction of AHL with human colon cancer epithelial HT-29 cells and colon cancer tissues. METHODS: Cell viability was determined by MTT [3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide] assay, while cell surface binding, apoptosis by Annexin-V-PI assay and ROS production using DCFDA [2',7' - dichlorofluorescindiacetate] kit method were analysed by flowcytometry, immunohistochemistry was performed using biotinylated AHL, protein purification by affinity chromatography using asialofetuin-coupled Sepharose -4B column. RESULTS: AHL strongly binds to HT-29 cells with a Mean Fluorescence Intensity of 12.4, which could be blocked by competing glycoprotein asialofetuin. AHL inhibits HT-29 cell growth in a dose and time-dependent manner with IC50 of 2.5 µg/mL and differentially binds to human normal and cancerous tissues. AHL induces apoptosis and slight necrosis in HT-29 cells with an increase in the early apoptotic population of 25.1 and 36% for 24 h and 48 h respectively and necrotic population of 1.5 and 4.6% at 24 h and 48 h respectively as revealed by Annexin-V-PI assay. AHL induces the release of Reactive Oxygen Species in HT-29 cells in a dose-dependent manner. CONCLUSION: To the best of knowledge, this is the first report on lectin from Adenia hondala which is not a RIP with apoptotic and necrotic effects. These findings support the promising potential of AHL in cancer research.

In Vitro Phytotherapeutic Properties of Aqueous Extracted Adenia viridiflora Craib. towards Civilization Diseases.

Adenia viridiflora Craib. is an indigenous edible plant that became an endangered species due to limited consumption of the local population with unknown reproduction and growth conditions. The plant is used as a traditional herb; however, its health applications lack scientific-based evidence. A. viridiflora Craib. plant parts (old leaves and young shoots) from four areas as Kamphaeng Phet (KP), Muang Nakhon Ratchasima (MN), Pakchong Nakhon Ratchasima (PN), and Uthai Thani (UT) origins were investigated for phenolic compositions and in vitro health properties through the inhibition of key enzymes relevant to obesity (lipase), diabetes (α-glucosidase and dipeptidyl peptidase-IV), Alzheimer's disease (cholinesterases and ß-secretase), and hypertension (angiotensin-converting enzyme). Phenolics including p-coumaric acid, sinapic acid, naringenin, and apigenin were detected in old leaves and young shoots in all plant origins. Old leaves exhibited higher total phenolic contents (TPCs) and total flavonoid contents (TFCs), leading to higher enzyme inhibitory activities than young shoots. Besides, PN and MN with higher TPCs and TFCs tended to exhibit greater enzyme inhibitory activities than others. These results will be useful to promote this plant as a healthy food with valuable medicinal capacities to support its consumption and agricultural stimulation, leading to sustainable conservation of this endangered species.

Purification, characterization and fine sugar specificity of a N-Acetylgalactosamine specific lectin from Adenia hondala.

Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC50 of 4.8 µg/ml. AHL can be explored for its clinical potential.

Role for monoaminergic systems in the antidepressant and anxiolytic properties of the hydroethanolic leaf extract from Adenia cissampeloides.

BACKGROUND: Adenia cissampeloides (Planch ex. Hook) Harms (Passifloraceae) leaf infusion is used in traditional African medicine as a stimulant to treat depression and insanity. Thus, this study investigates antidepressant and anxiolytic activities of the hydroethanol leaf extract of Adenia cissampeloides (ACE) in mice. METHODS: ACE (50-200 mg/kg, p.o.) was administered to mice 1 h before behavioral studies; the forced swimming test (FST), tail suspension test (TST), elevated-plus maze test (EPM) hole-board test (HBT) and open field test (OFT). In addition, the probable mechanisms of antidepressant- and anxiolytic-like actions of ACE were also investigated. RESULTS: ACE (100 and 200 mg/kg) produced significant (p<0.01) reduction in immobility, along with a significant increase in swimming activity (75.20%) and climbing (190.00%), respectively, similar to anti-immobility effect of imipramine in the FST. Also, in TST, ACE (100 and 200 mg/kg) treatment significantly (p<0.01) reduced the immobility time by 35.60%, and 35.27%, respectively, which was similar to anti-immobility effect of fluoxetine (32.50%). However, the antidepressant-like effect produced by ACE was prevented (p<0.01) by yohimbine (α2-adrenoceptor antagonist), or sulpiride (dopamine D2 receptor antagonist) pretreatment. ACE (50 and 100 mg/kg) treatment (p<0.01) increased number (41.67%) and duration of head-dips (52.27%) in HBT. Similarly, ACE (50-200 mg/kg) increased duration of open arm entries (p<0.001) in EPM. However, this effect was reversed (p<0.001) by pretreatment of mice with cyproheptadine (5-HT2 receptor antagonist) (60.87%). CONCLUSIONS: Findings from these studies revealed antidepressant-like effect of ACE mediated through interaction with dopamine D2- receptor or α2-adrenoceptor. Also an anxiolytic-like effect through interaction with 5-HT2 receptors.

Ethnobotany, phytochemistry, and bioactivity of the genus Turnera (Passifloraceae) with a focus on damiana--Turnera diffusa.

ETHNOPHARMACOLOGICAL RELEVANCE: Half a dozen of the currently accepted 135 Turnera species are used in traditional medicine, most notably Turnera diffusa Willd. ex Schult. which is one of the most highly appreciated plant aphrodisiacs. Other traditional uses of Turnera L. species include the treatment of anaemia, bronchitis, cough, diabetes, fever, fungal disease, gastrointestinal complaints, pain, pulmonary and respiratory diseases, skin disorders, and women׳s health problems. Additionally, Turnera species are used as abortives, expectorants, and laxatives. PHYTOCHEMISTRY: Flavonoids (22 different compounds), maltol glucoside, phenolics, cyanogenic glycosides (7 different compounds), monoterpenoids, sesquiterpenoids, triterpenoids, the polyterpene ficaprenol-11, fatty acids, and caffeine have been found in the genus Turnera. BIOACTIVITY: Bioactivities experimentally proven for members of the genus Turnera encompass antianxiety, antiaromatase, antibacterial including antimycobacterial, antidiabetic, antioxidant, adapatogenic, antiobesity, antispasmodic, cytotoxic, gastroprotective, hepatoprotective, and aphrodisiac activities. Most of these activities have so far been investigated only in chemical, cell based, or animal assays. In contrast, the antiobesity activity was also investigated in a study on healthy human subjects and with a herbal preparation containing among other ingredients Turnera diffusa leaves. Moreover, the enhancement of female sexual function was assessed in humans; again the product contained besides Turnera diffusa other potentially bioactive ingredients. However, with only few exceptions, most of the traditional uses and the experimentally verified bioactivities can currently not be related to a particular compound or compound class. A notable exception is the flavonoid apigenin, which was identified animal experiments as the antinociceptive principle of Turnera diffusa. CONCLUSION: In this review, the current knowledge on ethnobotanical uses of members of the genus Turnera, the secondary metabolites reported from Turnera, and experimentally documented bioactivities from Turnera extracts and pure compounds derived from Turnera extracts are compiled. Moreover, some of the most interesting avenues for future research projects are being discussed briefly. These include in particular the aphrodisiac activity of Turnera diffusa and the antibiotic activity potentiating effect of Turnera ulmifolia L. against aminoglycoside resistant bacteria.

Antihyperglycaemic and antioxidant effects of Adenia lobata Engl. (Passifloraceae) in streptozotocin-induced diabetic rats.

The antihyperglycaemic and antioxidant activities of a Ghanaian medicinal plant namely Adenia lobata Engl (Passifloraceae), used to treat diabetes mellitus in traditional medicine, was investigated. The dried stem powder of A. lobata was successively extracted by Soxhlet with petroleum ether and 70% ethanol to obtain the crude petroleum ether (PEAL: yield =1.1 w/w %) and ethanol (EEAL: yield = 5.4 w/w %) extracts. The extracts were assessed for their antihyperglycaemic and antioxidant activities. The antihyperglycaemic activity of PEAL and EEAL were determined in streptozotocin-induced diabetic rats (70 mg/kg body weight). Five groups of diabetic rats were given 150, 300 and 600 mg/kg body weight of PEAL and EEAL orally once daily for 20 days. Glibenclamide (5 mg/kg body weight) was used as positive control while distilled water (5 ml) acted as the normal diabetic control. The blood glucose levels were monitored initially for 6 hours and subsequently over 24 days. Both extracts exhibited statistically significant (p< 0.001) antihyperglycaemic activity throughout the study period, with EEAL showing the greatest activity. The antioxidant properties of the petroleum ether and ethanol extracts of A. lobata (PEAL and EEAL) were evaluated using five assays; total phenolic content, total antioxidant capacity, reducing power, DPPH scavenging effect and lipid peroxidation activity. In all these assays, the antioxidant properties increased with increasing concentration of the extracts.

Respuesta in vitro de diferentes biotipos y explantos de Passiflora caerulea L / In vitro response of different biotypes and explants of Passiflora caerulea L

Passiflora caerulea L., al igual que otras especies de la familia Passifloraceae, es utilizada en la medicina popular por sus propiedades antiespasmódicas y para el tratamiento de la ansiedad, el insomnio y el nerviosismo. La belleza de sus flores les otorga valor ornamental, mientras que sus frutos son apreciados por su importancia alimenticia. Se evaluó la respuesta in vitro de diferentes explantos y tres biotipos de P. caerulea: Corral de Bustos (provincia de Córdoba), Zavalla (provincia de Santa Fe) y Neuquén (provincia de Neuquén). Se utilizaron dos tipos de explantos: entrenudos y segmentos nodales, y como medio de cultivo Murashige y Skoog (1962) (MS), suplementado con vitaminas de Gamborg (1976) y 1 mg/L-1 de benciladenina (BA). Las respuestas fueron diferentes según el genotipo y el explanto. Los entrenudos ubicados tanto horizontal como verticalmente en medio de cultivo generaron callos como única respuesta. El biotipo de Neuquén mostró los mayores porcentajes de segmentos nodales con brotes. A través de estudios histológicos se determinó que en medio de cultivo MS con 1 mg/L-1 de BA, los segmentos nodales de P. caerulea originan brotes a partir de las yemas axilares preformadas y raíces que parten de callos en la base de los mismos. En iguales condiciones, los entrenudos originan callo como única respuesta.

As other species of the Passifloraceae family, Passiflora caerulea L. is used in popular medicine for its antispasmodic properties and as a remedy for anxiety, insomnia and nervousness. It is also highly prized for the ornamental value of its beautiful flowers, as well as for the nutritional importance of its fruits. The in vitro response of different explants and three biotypes of P. caerulea: the Corral de Bustos (Province of Córdoba), the Zavalla (Province of Santa Fe) and the Neuquén (Province of Neuquén) genotypes, was evaluated using two types of explants: internodes and nodal segments on Murashige and Skoog (1962) (MS) culture medium supplemented with Gamborg’s vitamins (1976) and 1 mg.L-1 of benzyladenine (BA). There were different responses depending on the genotype and the explant. The internodes placed both horizontally and vertically in the culture medium produced callus as sole response. The Neuquén biotype showed the highest percentages of nodal segments with shoots. Histological tests allowed to establish that in MS culture medium with 1 mg.L-1 of BA, the nodal segments of P. caerulea produce shoots from the preformed axillary buds and roots that develop from the callus situated on its base. Under similar conditions, the internodes produce callus as sole response.

Anaesthetic effects of Adenia gummifera distillates on Apis mellifera (Honeybee).

The anaesthetic activities of steam distillates of Adenia gummifera stem on Apis mellifera were evaluated by a diffusion method. Live, direct and fractional (61-80 degrees C fraction) distillates had greater anaesthetic effect while vacuum distillates were mild. The anaesthetic activity significantly increased with concentration up to 6% (v/v), and then it levelled off, while excessive exposure was lethal. The number of bees in a given volume had no significant effect on anaesthetic activity but container volume (F(cal) = 66.4; F(3,8) = 4.07) and bee-distillate distance (F(cal) = 31.0; F(2,6) = 5.14) did, suggesting the rate of diffusion of active component could be the determining factor. The active component is likely to contain amines and the rest halogenated alkane.

In vitro and in vivo toxicity of type 2 ribosome-inactivating proteins lanceolin and stenodactylin on glial and neuronal cells.

Lanceolin and stenodactylin, new type 2 ribosome-inactivating proteins (RIPs) from Adenia plants were recently isolated and their high cytotoxicity was described. Present experiments were performed to investigate the effect of these toxins on neural cells in culture and their in vivo retrograde transport and neurotoxicity in the central nervous system. The concentrations of lanceolin and stenodactylin inhibiting by 50% protein synthesis were in the 10(-11) and 10(-12) (cerebellar granule neurons), 10(-12) and 10(-13) (astrocytes), and 10(-13) (microglia) molar range, respectively. Both RIPs resulted toxic for glial cells in culture by MTT test, killing 50% of microglia, the most sensitive cell type, at concentrations around 10(-14)M. Stenodactylin was highly neurotoxic in vivo, when injected intracerebrally, and was retrogradely transported through axons projecting to the injected region. Stereotaxic injection of 1.3 ng toxin into the left dorsal hippocampus resulted in loss of cholinergic neurons in the ipsilateral medial septal nucleus, where cell bodies of neurons providing cholinergic input to the hippocampus are located. The retrograde transport of RIPs along neurons allows to perform experiments of target-selective lesioning, and can be exploited also to perform specific experiments of immunolesioning of selected neuronal populations.

Ribosome-inactivating proteins and other lectins from Adenia (Passifloraceae).

The caudices of 10 Adenia species contain galactose-binding lectins that were purified by affinity chromatography. All lectins but three agglutinate human erythrocytes. Six lectins consist of two unequal chains, which can be separated by reduction, and inhibit protein synthesis both by a rabbit reticulocyte lysate and by HeLa and Raji cells. The lectins from A. goetzii, A. lanceolata and A. stenodactyla had the highest cytotoxicity, inhibiting cell protein synthesis with IC50s (concentration inhibiting by 50%) below 0.1 ng/ml, and deadenylate DNA, thus being type 2 ribosome-inactivating proteins.

Cyanogenic allosides and glucosides from Passiflora edulis and Carica papaya.

Leaf and stem material of Passiflora edulis (Passifloraceae) contains the new cyanogenic glycosides (2R)-beta-D-allopyranosyloxy-2-phenylacetonitrile (1a) and (2S)-beta-D-allopyranosyloxy-2-phenylacetonitrile (1b), along with smaller amounts of (2R)-prunasin (2a), sambunigrin (2b), and the alloside of benzyl alcohol (4); the major cyanogens of the fruits are (2R)-prunasin (2a) and (2S)-sambunigrin (2b). The major cyanogenic glycoside of Carica papaya (Caricaceae) is 2a; only small amounts of 2b also are present. We were not able to confirm the presence of a cyclopentenoid cyanogenic glycoside, tetraphyllin B, in Carica papaya leaf and stem materials. In detailed 1H NMR studies of 1a/b and 2a/b, differences in higher order effects in glucosides and allosides proved to be valuable for assignment of structures in this series. The diagnostic chemical shifts of cyanogenic methine and anomeric protons in 1a/b are sensitive to anisotropic environmental effects. The assignment of C-2 stereochemistry of 1a/b was made in analogy to previous assignments in the glucoside series and was supported by GLC analysis of the TMS ethers.