Biosynthesis of silver nanoparticle from flower extract of Dillenia indica and its efficacy as antibacterial and antioxidant.

Dillenia indica is a medicinal tree of the Dilleniaceae and its flower extract was used for the synthesis of silver nanoparticle (AgNPs). The optimal conditions for AgNPs synthesis were as such: 2 mM AgNO3, pH 4.5 and 48-h reaction time. The characteristic band of AgNPs was observed at the wavelength of 435 nm by UV-visible spectroscopic study. Fourier-transform infrared (FTIR) analysis depicted the involvement of several functional groups of plant extracts in the synthesis of AgNPs. Nanoparticles were mostly spherical shaped and uniformly distributed, when observation was made by Transmission electron microscopy (TEM). Energy Dispersive X-Ray (EDX) showed absorption peak approximately at 3 keV thus confirmed the presence of silver metal in AgNP. X-ray diffraction (XRD) investigation and selected area electron diffraction (SAED) patterns showed the crystalline nature of the AgNPs. Dynamic light scattering (DLS) analysis exhibited average size of the nanoparticles as 50.17 nm with a polydispersity index (PDI) value of 0.298. The zeta potential of nanoparticles was observed as -24.9 mV. To assess antibacterial activity, both AgNPs alone or its combination with the antibiotic were tried against six pathogenic bacteria. The combination of AgNPs with antibiotic was maximum effective against Shigella boydii (16.07 ± 0.35) and Klebsiella pneumoniae (15.03 ± 0.20). AgNPs alone showed maximum inhibition for both Gram-positive bacteria: methicillin-resistant Staphylococcus aureus (19.97 ± 0.20 mm) and Enterococcus faecium (19.80 ± 0.15 mm). Maximum inhibition of Enterobactor cloacae and Pseudomonas aeruginosa was observed by antibiotic taken alone. Evaluation through 2,2-diphenyl-1-picrylhydrazyl (DPPH) and DNA nicking assays demonstrated the antioxidant capabilities of the nanoparticles.

Antinociceptive Effect of Dillenia indica (Linn.) Mediated by Opioid and Cannabinoid Systems: Pharmacological and Chemical Studies.

Dillenia indica (Linn.) has been reported by several biological activities, including anti-inflammatory, antioxidant, antidiabetic, anti-hyperglycemic, antiproliferative, antimutagenic, anticholinesterase, and antimicrobial. In Brazilian traditional medicine, the fruits of D. indica have been used to treat general topical pain and inflammation, but with no scientific validation. Thus, aiming to study its chemical constitution and antinociceptive properties, the crude extract (CE) and fractions obtained from the fruits of D. indica were submitted to an in vivo pharmacological evaluation and a dereplication study by LC-MS/MS analysis, assisted by the Global Natural Product Social Molecular Networking (GNPS). The oral antinociceptive activity of the fruits of D. indica and the possible participation of the opioid and cannabinoid systems were demonstrated in the formalin-induced nociception model. The chemical dereplication study led us to identify several known chemical constituents, including flavonoids, such as caffeoylmalic acid, naringenin, quercetin, and kaempferol. According to literature data, our results are compatible with significant antinociceptive and anti-inflammatory activities. Therefore, the flavonoid constituents of the fruits of D. indica are probably responsible for its antioxidant, anti-inflammatory, and antinociceptive effects mediated by both opioid and cannabinoid systems, confirming its folk use in the treatment and relief of pain.

Exploration of the chemical constituents and its antioxidant, antibacterial activities of endophytic fungi isolated from the medicinal plant Dillenia indica.

Assam, India being the pool for ethnomedicinal plants harbors diverse endophytic fungi constituting major bioactive metabolites. The present study was designed to screen the antioxidant, antibacterial activities along with the chemical constituents of the endophytic fungi isolated from the fruits of Dillenia indica (commonly known as Otenga in Assam). Screening of such metabolic compounds and their antioxidant, antibacterial activities can have tremendous potential in suppressing certain diseases. Agar well diffusion method has been used to carry out the antibacterial assay against three pathogenic bacteria two gram positive [Bacillus subtilis (MTCC No. 441); Staphylococcus aureus (MTCC No. 740)] and one gram negative [Escherichia coli (MTCC No. 739)]. Aspergillus fumigatus of ethyl acetate extract showed a prominent activity against Staphylococcus aureus followed by Aspergillus flavus and Aspergillus niger. Antioxidants have the potential to neutralize and inhibit the action of free radicals. The highest scavenging activity was exhibited by ethyl acetate extract of Aspergillus fumigatus in DPPH assay. Furthermore, the phytochemical screening revealed the presence of flavonoids, alkaloids, terpenoids and saponins. Result showed that ethyl acetate extract of Aspergillus fumigatus showed the highest phenolic content (236.81 ± 0.2 mg.g-1) and least was shown by Aspergillus flavus (92.12 ± 1.4 mg.g-1). Total flavonoids content for Aspergillus fumigatus (39.08 ± 0.2 mg.g-1) was found to be highest compared to other isolates. Molecular identification of the endophytic fungus showing highest activity was done based on 18S rRNA. The sequenced was submitted in Genbank with accession number MH540721 showing high similarities with Aspergillus fumigatus strain 3,162,954. A. fumigatus strain is subjected to GC/MS analysis that revealed the chemical constituents 2-isopropyl-5-methyl-1-heptanol, dodecane, 1-fluoro-pentanoic acid, 2-ethylhexyl ester, 1-octanol, 2-butyl-1-dodecanol. Thus, the present work reveals that endophytic fungi colonizing in ethnomedicinal plant Dillenia indica could be a promising source for antioxidant and antibacterial activity. Further work is needed to add value in various therapeutic and pharmaceutical fields.

Anti-MRSA and clot lysis activities of Pestalotiopsis microspora isolated from Dillenia pentagyna Roxb.

There is an urgent call to search for novel natural compounds against developing multidrug-resistant microorganisms. The present work focuses on the characterization of a plant-associated fungus having bioactivity against methicillin-resistant Staphylococcus aureus (MRSA) strains. A fungal strain P31 was isolated from bark of Dillenia pentagyna and identified as Pestalotiopsis microspora. The maximum anti-MRSA activity was observed from extract of P31 grown in sabouraud dextrose broth. The minimum inhibitory concentrations (MIC) values of P31 extract were 14 µg/ml for methicillin-sensitive S. aureus (MSSA) and 32 µg/ml for MRSA strain, respectively. A crude P31 extract showed strong bactericidal activity by killing all treated MRSA cells within 24 h of treatment at their respective MIC value. A scanning electron microscopic study visualized morphological damage of MRSA cells. The membrane permeability of P31 extract-treated MRSA cells gradually increased which caused release of internal cytoplasmic nucleic acids, proteins and potassium ions (K+ ) from cells suggesting cell lysis or leakage from cells. A very low concentration of P31 extract was able to inhibit biofilm formed by MRSA cells. Thin layer chromatographic separation followed by gas chromatography-mass spectrometry analysis of the P31 extract revealed a number of antimicrobial compounds along with an anti-MRSA compound 2,4-di-tert-butylphenol. In addition, the P31 extract also showed in-vitro human blood clot lysis activity at various concentrations. The clot lysis activity of P31 extract was found maximum at 500 µg/ml. These findings suggest that fungal isolate P31 has potential as a source of anti-MRSA compounds useful in staph infections.

Anti-Zika Virus Activity and Isolation of Flavonoids from Ethanol Extracts of Curatella americana L. Leaves.

The ethnomedicinal plant Curatella americana L. (Dilleniaceae) is a common shrub in the Brazilian Cerrado, whose ethanolic extract showed significant in vitro anti-Zika virus activity by the MTT colorimetric method. Currently, there is no drug in clinical use specifically for the treatment of this virus; therefore, in this work, the antiviral and cytotoxic properties of the ethanolic extract, fractions, and compounds were evaluated. The ethanolic extract of the leaves showed no cytotoxicity for the human MRC-5 cell and was moderately cytotoxic for the Vero cell (CC50 161.5 ± 2.01 µg/mL). This extract inhibited the Zika virus multiplication cycle with an EC50 of 85.2 ± 1.65 µg/mL. This extract was fractionated using the liquid-liquid partition technique, and the ethyl acetate fraction showed significant activity against the Zika virus with an EC50 of 40.7 ± 2.33 µg/mL. From the ethyl acetate fraction, the flavonoids quercetin-3-O-hexosylgallate (1), quercetin-3-O-glucoside (2), and quercetin (5) were isolated, and in addition to these compounds, a mixture of quercetin-3-O-rhamnoside (3) and quercetin-3-O-arabinoside (4) was also obtained. The isolated compounds quercetin and quercetin-3-O-hexosylgallate inhibited the viral cytopathic effect at an EC50 of 18.6 ± 2.8 and 152.8 ± 2.0, respectively. Additionally, analyses by liquid chromatography coupled to a mass spectrometer allowed the identification of another 24 minor phenolic constituents present in the ethanolic extract and in the ethyl acetate fraction of this species.

Seasonal variation and tissues specificity of endophytic fungi of Dillenia indica L. and their extracellular enzymatic activity.

Endophytes are microbes that live inside the tissues of plants without causing any disease. Many of these belonging to fungi have been exploited earlier for their biological activities. This study focused on the exploration and characterization of culturable endophytic fungi inhabiting the medicinal plant Dillenia indica L. during four different seasons (summer, monsoon, autumn, and winter) from 2018 to 2019. A total of 2360 segments from different parts (leaves, fruits, and stem) were screened to isolate endophytic fungi. During the study, 25 species of fungi belonging to 20 genera were isolated from the selected plant. The identification of these fungi was validated at morphological, microscopic, and molecular levels. Results indicate the plant has the highest affinity for Daldinia eschscholtzii, followed by Colletotrichum gloeosporioides and Cladosporium cladosporioides. Further, the percent frequency was highest in leaves, followed by stem and fruits. The results were further supported by a similar trend of colonization rate for different plant parts. The monsoon season had the highest number of isolates (312), followed by summer (208), winter (164), and autumn (114). Species diversity was highest during the monsoon season and lowest during the winter. These fungi also produce amylase, lipase, protease, asparaginase, cellulase, and ligninolytic enzymes. This study focused only on culturable fungal endophytes, yet the scope can be extended for other non-culturable microbes and their interaction by using high-throughput genomics and novel next-generation sequencing (NGS) tools. The results indicate that Dillenia indica L. harbors novel endophytic fungi having industrial applications.

Toxicological evaluation of ethanolic extract of leaves from Doliocarpus dentatus in Swiss mice.

Doliocarpus dentatus (Dilleniaceae) has been used in folk medicine to treat inflammation and pain; however, studies evaluating its toxicity potential, as well as its effects on anxiety and depression, are scarce. This study investigated the toxicological profile of an ethanolic extract from leaves of D. dentatus (EEDd), and its effects on anxiety and depression models in mice. Male and female mice received either a single dose (500, 1000 or 2000 mg/kg) or repeated doses (75, 150 or 300 mg/kg) of EEDd by oral gavage. During the subacute toxicity assay, behavioral tests were performed on days 4, 14, 21 and 28. No evidence of toxicity was observed in the animals in both acute and subacute tests. However, males treated with the highest dose presented a reduction in the absolute weight of the kidney, an elevation in the AST levels, in addition to an alteration in the urea levels. The treatment did not affect other biochemical parameters, and did not induce any depressive-like behavior. EEDd exhibited low toxicity after single and repeated exposures. Since some analyzed parameters were compromised, further toxicity studies should be carried out.

Root positioning and trait shifts in Hibbertia racemosa as dependent on its neighbour's nutrient-acquisition strategy.

Nutrient-poor ecosystems globally exhibit high plant diversity. One mechanism enabling the co-existence of species in such ecosystems is facilitation among plants with contrasting nutrient-acquisition strategies. The ecophysiological processes underlying these interactions remain poorly understood. We hypothesized that root positioning plays a role between sympatric species in nutrient-poor vegetation. We investigated how the growth traits of the focal mycorrhizal non-cluster-rooted Hibbertia racemosa change when grown in proximity of non-mycorrhizal Banksia attenuata, which produces cluster roots that increase nutrient availability, compared with growth with conspecifics. Focal plants were placed in the centre of rhizoboxes, and biomass allocation, root system architecture, specific root length (SRL), and leaf nutrient concentration were assessed. When grown with B. attenuata, focal plants decreased root investment, increased root growth towards B. attenuata, and positioned their roots near B. attenuata cluster roots. SRL was greater, and the degree of localized root investment correlated positively with B. attenuata cluster-root biomass. Total nutrient contents in the focal individuals were greater when grown with B. attenuata. Focal plants directed their root growth towards the putatively facilitating neighbour's cluster roots, modifying root traits and investment. Preferential root positioning and root morphological traits play important roles in positive plant-plant interactions.

Antibacterial and synergistic activity of 6ß-hydroxy-3-oxolup-20(29)-en-28-oic acid (6ß-hydroxy betunolic acid) isolated from Schumacheria castaneifolia vahl.

Multi- drug resistant microbial pathogens are a serious global health problem and thus new antibacterial agents, which are effective both alone and in combination with traditional antibiotics, are urgently needed. Hence, the objective of the present study was to investigate the antibacterial activity of 6ß-hydroxy-3-oxolup-20(29)-en-28-oic acid (6ß-hydroxy betunolic acid) isolated from the bark of Schumacheria castaneifolia and its effect when combined with oxacillin. Antibacterial potential of 6ß-hydroxy betunolic acid alone was performed using broth micro dilution assay against sixteen bacterial strains which included eight standard strains [Staphylococcus aureus (ATCC 29213 and ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 35218 and ATCC 25922), carbapenemase producing Kebsiella pneumonia (ATCC BAA 1705), carbapenemase non-producing K. pneumonia (ATCC BAA 1706) and Pseudomonas aeruginosa (ATCC 27853)] and four strains each of clinically isolated meropenem resistant Acinetobactor sp. and methicillin resistant S. aureus (MRSA) which were included in the urgent threat list and serious threat list, respectively in 2019 by the Centers for Disease Control and Prevention in the United States. Its effect when combined with oxacillin was tested against S. aureus (ATCC 29213) and MRSAs using a checkerboard dilution method. The results indicated that 6ß-hydroxy betunolic acid had antibacterial activity against the tested Gram positive organisms with MICs ranging from 16 to 32 mg L-1 (MIC of oxacillin and meropenem ranged from 0.25-16 and 0.03-128 mg L-1 respectively). The high MIC values (>1024 mg L-1) of 6ß-hydroxy betunolic acid against Gram negative strains indicated a likely lack of activity. Further, 6ß-hydroxy betunolic acid exhibited synergistic effect with oxacillin against Staphylococcus aureus (0.49) and showed an additive effect against all the tested MRSAs. The present study suggested that the antibacterial activity of the 6ß-hydroxy betunolic acid is restricted to Staphylococcus isolates and possibly Enterococcus faecalis. Further testing on different types of Gram positives and identification of the exact mechanism of action would be of importance.

The design of ibuprofen-loaded microbeads using polymers obtained from Xanthosoma sagittifolium and Dillenia indica.

BACKGROUND: Ibuprofen is used both for acute and chronic disorders, such as ankylosing spondylitis, osteoarthritis and rheumatoid arthritis; however, ibuprofen causes gastrointestinal disturbances. Therefore, it would be desirable to design it as a sustained-release preparation. OBJECTIVES: To design ibuprofen microbeads using polymers obtained from Xanthosoma sagittifolium starch and Dillenia indica mucilage to provide sustained-release delivery of ibuprofen. MATERIAL AND METHODS: The polymers were extracted using standard methods and characterized by their material, physicochemical, elemental, and rheological profiles. Microbeads loaded with ibuprofen were prepared using the ionotropic gelation method utilizing blends of the polymers and sodium alginate. The microbeads were evaluated using particle shape, particle size, swelling index, entrapment efficiency, and release assays. RESULTS: The results showed that the polymers have distinct material and physicochemical properties unique to their botanical sources. The microbeads were spherical and free-flowing, and they rolled without friction. The swelling properties ranged from 47.62 ±2.74% to 79.49 ±3.66%. The particle size of the microbeads ranged from 88.14 ±68.57 µm to 214.90 ±66.95 µm, while the encapsulation efficiencies ranged from 20.67 ±4.66% to 83.61 ±6.35%. The dissolution times suggested that the concentration of the natural polymers in the bead formulation could be used to modulate the dissolution properties. Generally, formulations containing the mucilage yielded higher dissolution times than those containing the starch. The kinetics of drug release from the microbeads containing the polymer blends generally fitted the Korsmeyer-Peppas model. The highest similarity was found between formulations C6 and D4 with f2 of 81.07. CONCLUSIONS: The microbeads prepared with polymers obtained from Xanthosoma and Dillenia showed acceptable physicochemical properties, dependent upon polymer type, blend and concentration.

Ethanolic extract of leaf of Dillenia pentagyna reduces in-vitro cell migration and induces intrinsic pathway of apoptosis via downregulation of NF-κß in human NSCLC A549 cells.

Despite the advancement of the pharmaceutical industry, medicinal plants are still a reliable source of traditional medicines to cure a number of diseases. Various parts of Dillenia pentagyna are used in traditional medicine in India for treatment of various disorders including cancers, but detailed mechanisms are still unknown. Dried leaves of D. pentagyna were extracted with ethanol and termed as an ethanolic extract of leaves of D. pentagyna (EELDP). Our aim was to elucidate the role of EELDP in in-vitro cell migration and apoptosis in highly metastatic human lung adenocarcinoma A549 cells. We measured cell viability and in-vitro cell migration in three different human cancer cells A549, HeLa and U2OS treated with EELDP (0-0.6 mg/mL). However, A549 cells showed higher sensitivity to EELDP treatment. Hence we studied several key markers of metastasis and apoptosis pathway in A549 cells treated with EELDP. EELDP treatment significantly reduced in-vitro cell migration, wound healing, expression and activity of MMP-2, MMP-9 via reduction of nuclear factor kappa Beta (NF-κß). EELDP also reduced vimentin, N-cadherin and increased claudin-1. The intrinsic pathway of apoptosis was triggered by EELDP via the NF-κß pathway through the increase of the Bax to Bcl2 ratio, leading to the fall of mitochondrial membrane potential and subsequently induced release of cytochrome c, activation of caspase-3 followed by nuclear fragmentation in A549 cells. Furthermore, we observed change of a few markers of metastasis and apoptosis in other two cell types HeLa and U2OS treated with EELDP. These data implicate that the effect of EELDP is not cell-specific. Since only 0.1 mg/mL EELDP significantly reduces in-vitro cell migration and increases apoptosis, the active compound(s) present in EELDP is very much potent to control highly metastatic cancer.

An overview of inventories of gall-inducing insects in Brazil: looking for patterns and identifying knowledge gaps.

We compiled published Brazilian gall-inducing insect inventories aiming to understand trends and biases in this field research and to investigate the factors that potentially explain the diversity of gall-inducing insects among different sampling sites. A total of 51 studies with gall-inducing insect inventories were compiled for Brazil, which sampled 151 sites in 88 municipalities, 13 states and five regions. The number of papers published on gall-inducing insects per year has increased over the last 30 years, being Cecidomyiidae (Diptera) the main galling taxon, Fabaceae the main host-plant family and Protium heptaphyllum (Burseraceae) the most important super-host species in these inventories. We found a great bias in the geographical distribution of Brazilian inventories, with the majority of studies in the Southeast region, and Atlantic Forest and Cerrado biomes. The total richness of gall-inducing insects differed significantly among regions and biomes, with higher gall richnesses being recorded in the North region and Amazon biome. However, Brazilian regions and biomes did not vary in richness of gall-inducing insect morphotypes per plant species. According our results, sampling by cecidologists in less studied regions of Brazil is needed, particularly in the North and South regions and subsampled biomes such as the Amazon, Pampas and Pantanal.

The phytochemical and anti-inflammatory studies of Dillenia suffruticosa leaves.

The Dillenia suffruticosa leaves (Dilleniaceae), a folk medicine recommended in Southeast Asia for treating inflammation, were phytochemically studied for the first time and assessed for suppression of λ-carrageenan-induced paw oedema in rats. The crude methanolic extract orally administered at 5,000 mg/kg, displayed no toxicity and at 250 to 1,000 mg/kg significantly suppressed the paw oedema. Two-isolated triterpenoids, betulinic acid (1) and koetjapic acid (2) orally administered at 50 mg/kg, significantly reduced the paw oedema, (p < 0.001) and (p < 0.005) at the fourth h onwards to 47.36% ± 2.23 and 53.43% ± 7.09, respectively, from 95.90% ± 6.88 oedema induced by λ-carrageenan alone. 1 and the isolated flavonoids of vitexin (3), tiliroside (4), and kaempferol (5), displayed moderately more of cyclooxygenase (COX)-2 than COX-1 enzyme inhibition, whereas 2 was slightly more inhibition of COX-1. The in silico molecular docking studies provided support to the in vitro COX studies that the isolated compounds formed H-bonding with the amino acid residues at the COX-2 catalytic sites. The triterpenoids were bound to the peroxidase, possibly inhibiting the peroxidase reaction, whereas the flavonoids interacted more at the cyclooxygenase, resembling celecoxib, therefore providing evidences that these compounds were responsible for the anti-inflammatory properties of D. suffruticosa.

Coagulation/flocculation of textile effluent using a natural coagulant extracted from Dillenia indica.

The aim of this study was to assess the efficiency of mucilage extracted from the fruit of Dillenia indica for enhancing coagulation in the treatment of textile effluent. The mucilage extraction was carried out in water at room temperature. The pH, concentration of coagulant FeCl3.6H2O, and concentration of mucilage solution were optimized with star-type central composite design (CCD). We were able to analyze the synergistic effects between the FeCl3.6H2O and mucilage concentrations: the process of coagulation/flocculation (CF) for chemical oxygen demand (COD) removal was more efficient at a low chemical coagulant concentration (8.00 mg L-1) and a higher natural coagulant (NC) concentration (15.00 mg L-1). This demonstrated the potential of this mucilage to treat textile effluents, with 67.66%% COD removal, 96.86% turbidity removal and 91.12% apparent color reduction. The characterization of the mucilage of Dillenia indica was done using Fourier transform infrared spectroscopy (FTIR) and solid-state cross-polarization magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS 13C NMR), and the signals obtained indicated the presence of polysaccharides, which are responsible for enhancing the CF process.

Flavonoids from Tetracera indica Merr. induce adipogenesis and exert glucose uptake activities in 3T3-L1 adipocyte cells.

BACKGROUND: Tetracera indica Merr. (Family: Dilleniaceae), known to the Malay as 'Mempelas paya', is one of the medicinal plants used in the treatment of diabetes in Malaysia. However, no proper scientific study has been carried out to verify the traditional claim of T. indica as an antidiabetic agent. Hence, the aims of the present study were to determine the in vitro antidiabetic potential of the T. indica stems ethanol extract, subfractions and isolated compounds. METHODS: The ethanol extract and its subfractions, and isolated compounds from T. indica stems were subjected to cytotoxicity test using MTT viability assay on 3T3-L1 pre-adipocytes. Then, the test groups were subjected to the in vitro antidiabetic investigation using 3T3-L1 pre-adipocytes and differentiated adipocytes to determine the insulin-like and insulin sensitizing activities. Rosiglitazone was used as a standard antidiabetic agent. All compounds were also subjected to fluorescence glucose (2-NBDG) uptake test on differentiated adipocytes. Test solutions were introduced to the cells in different safe concentrations as well as in different adipogenic cocktails, which were modified by the addition of compounds to be investigated and in the presence or absence of insulin. Isolation of bioactive compounds from the most effective subfraction (ethyl acetate) was performed through repeated silica gel and sephadex LH-20 column chromatographies and their structures were elucidated through 1H-and 13C-NMR spectroscopy. RESULTS: Four monoflavonoids, namely, wogonin, norwogonin, quercetin and techtochrysin were isolated from the T. indica stems ethanol extract. Wogonin, norwogonin and techtochrysin induced significant (P < 0.05) adipogenesis like insulin and enhanced adipogenesis like rosiglitazone. Wogonin and norwogonin also exhibited significant (P < 0.05) glucose uptake activity. CONCLUSION: The present study demonstrated that the flavonoids isolated from the T. indica stems possess antidiabetic potential revealing insulin-like and insulin-sensitizing effects which were significant among the compounds. This also rationalizes the traditional use of T. indica in the management of diabetes in Malaysia.

Quality comparison of elephant apple juices after high-pressure processing and thermal treatment.

BACKGROUND: In the present work, the effect of high pressure processing (HPP) on the quality parameters (pH, °Brix, total acidity, viscosity, colour, antioxidant activity, total phenols, total flavonoids, microbial flora, and sensory analysis) of elephant apple (Dillenia indica) juice was investigated. The juice samples were analysed periodically (0, 1, 2, 5, 10, 20, 30, 40, 50 and 60 days) during 60 days of storage period and results were compared with thermally processed as well as with untreated (fresh juice) samples. RESULTS: Slight variations had been observed in the quality parameters like pH, °Brix and total acidity. Other parameters like colour values, antioxidant activity, total phenols and total flavonoids were varied significantly (P < 0.05) in between the treated (HPP and thermal) and untreated juice samples. The microbial counts of the HPP treated samples were lower than the other samples. Sensory results also showed similar results to those of the other analyses that the treated samples were better for consumption rather than the untreated samples. The shelf-life of the HPP processed elephant apple juice was established as 60 days at 4 °C. CONCLUSION: This study showed that application of HPP effectively maintained quality attributes and extended shelf life of the elephant apple juice. It may be suggested that application of HPP could be considered for commercial application during storage and marketing. © 2016 Society of Chemical Industry.

Healing effect of Dillenia indica fruit extracts standardized to betulinic acid on ultraviolet radiation-induced psoriasis-like wounds in rats.

CONTEXT: Dillenia indica Linn. (Dilleniaceae) is traditionally used to treat skin inflammation. OBJECTIVE: This study evaluated the healing effect of Dillenia indica fruit extracts on induced psoriasis-like wounds in Wistar rats. MATERIALS AND METHODS: Extracts were standardized to betulinic acid, including an aqueous ethanolic extract (AEE), ethyl acetate extract (EAE) and petroleum ether extract. Effects against lipid peroxidation were assessed in vitro. Wounds were created at rat tails (n = 12). Topical treatments were applied once daily for 7 days (1 mL of AEE or EAE at 5 or 50 mg/mL). Maximal dose was defined by the extract solubility. A 10-fold lower dose was also tested. Positive and negative controls were treated with clobetasol (0.5 mg/mL) or excipient. Half of each group was euthanized for histology. The remaining animals were observed for 20 days for wound measurements. RESULTS: Yields of AEE and EAE were 4.3 and 0.7%, respectively. Betulinic acid concentrations in AEE and EAE were 4.6 and 107.6 mg/g. Extracts neutralized lipid peroxidation in vitro at 0.02 µg/mL, accelerating healing at 50 mg/mL. Complete healing in mice treated with AEE occurred 16 days after wound induction. This time was 14 and 12 days in mice treated with EAE and clobetasol. Compared to orthokeratosis, parakeratosis was reduced by AEE (25%), EAE (45%) and clobetasol (55%). EAE caused superior protection against biomolecules oxidation of skin compared to AEE. DISCUSSION AND CONCLUSION: EAE exhibited activity closer to that of clobetasol. Betulinic acid may be an active constituent, which should be assessed in future studies.

The Anti-Inflammatory Effects of the Methanolic Extract and Fractions from Davilla elliptica St. Hil. (Dilleniaceae) on Bothrops jararaca Envenomation.

Inflammation and haemorrhage are the main characteristics of tissue injury in botropic envenomation. Although some studies have shown that anti-venom prevents systemic reactions, it is not efficient in preventing tissue injury at the site of the bite. Therefore, this work was undertaken to investigate the anti-inflammatory effects of the methanolic extract and fractions from D. elliptica and to evaluate the role of matrix metalloproteinases (MMPs) in this process. Effects of the extract and fractions from D. elliptica were evaluated using a carrageenan-induced paw oedema model in rats, and leukocyte rolling was visualized by intravital. The quantification of MMPs activities (MMP-2 and MMP-9) extracted from the dermis of mice treated with extract and fractions alone or incubated with venom was determined by zymographic analyses. Our results show that intraperitoneal (i.p.) injection of fractions significantly reduced paw oedema after the carrageenan challenge. Treatment with the tannins fraction also resulted in considerable inhibition of the rolling of leukocytes and this fraction was able to decrease the activation of MMP-9. These results confirmed the anti-inflammatory activity of the methanolic extract and tannins fraction of D. elliptica and showed that the dermonecrosis properties of B. jararaca venom might be mediated through the inhibition of MMP-9 activity.

Inhibitory effect of triterpenoids from Dillenia serrata (Dilleniaceae) on prostaglandin E2 production and quantitative HPLC analysis of its koetjapic acid and betulinic acid contents.

The crude methanol extracts and fractions of the root and stem barks of Dillenia serrata Thunb. showed 64% to 73% inhibition on the production of prostaglandin E2 (PGE2) in lipopolysaccharide-induced human whole blood using a radioimmunoassay technique. Three triterpenoids isolated from the root bark of the plant, koetjapic (1), 3-oxoolean-12-en-30-oic (2), and betulinic (3) acids, exhibited significant concentration-dependent inhibitory effects on PGE2 production with IC50 values of 1.05, 1.54, and 2.59 µM, respectively, as compared with the positive control, indomethacin (IC50 = 0.45 µM). Quantification of compounds 1 and 3 in the methanol extracts and fractions were carried out by using a validated reversed-phase high performance liquid chromatography (RP-HPLC) method. The ethyl acetate fraction of the stem bark showed the highest content of both compound 1 (15.1%) and compound 3 (52.8%). The strong inhibition of the extracts and fractions on cyclooxygenase-2 (COX-2) enzymatic activity was due to the presence of their major constituents, especially koetjapic and betulinic acids.

Induction of cell cycle arrest and apoptosis in caspase-3 deficient MCF-7 cells by Dillenia suffruticosa root extract via multiple signalling pathways.

BACKGROUND: Dillenia suffruticosa root dichloromethane extract (DCM-DS) has been reported to exhibit strong cytotoxicity towards breast cancer cells. The present study was designed to investigate the cell cycle profile, mode of cell death and signalling pathways of DCM-DS-treated human caspase-3 deficient MCF-7 breast cancer cells. METHODS: Dillenia suffruticosa root was extracted by sequential solvent extraction. The anti-proliferative activity of DCM-DS was determined by using MTT assay. The mode of cell death was evaluated by using inverted light microscope and Annexin-V/PI-flow cytometry analysis. Cell cycle analysis and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry. MCF-7 cells were co-treated with antioxidants α-tocopherol and ascorbic acid to evaluate whether the cell death was mainly due to oxidative stress. GeXP-based multiplex system was employed to investigate the expression of apoptotic, growth and survival genes in MCF-7 cells. Western blot analysis was performed to confirm the expression of the genes. RESULTS: DCM-DS was cytotoxic to the MCF-7 cells in a time-and dose-dependent manner. The IC50 values of DCM-DS at 24, 48 and 72 hours were 20.3 ± 2.8, 17.8 ± 1.5 and 15.5 ± 0.5 µg/mL, respectively. Cell cycle analysis revealed that DCM-DS induced G0/G1 and G2/M phase cell cycle arrest in MCF-7 cells at low concentration (12.5 and 25 µg/mL) and high concentration (50 µg/mL), respectively. Although Annexin-V/PI-flow cytometry analysis has confirmed that DCM-DS induced apoptosis in MCF-7 cells, the distinct characteristics of apoptosis such as membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies were not observed under microscope. DCM-DS induced formation of ROS in MCF-7 cells. Nevertheless, co-treatment with antioxidants did not attenuate the cell death at low concentration of DCM-DS. The pro-apoptotic gene JNK was up-regulated whereby anti-apoptotic genes AKT1 and ERK1/2 were down-regulated in a dose-dependent manner. Western blot analysis has confirmed that DCM-DS significantly up-regulated the expression of pro-apoptotic JNK1, pJNK and down-regulated anti-apoptotic AKT1, ERK1 in MCF-7 cells. CONCLUSION: DCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via multiple signalling pathways. It shows the potential of DCM-DS to be developed to target the cancer cells with mutant caspase-3.