Grass pollen sensitization in dogs in Paraná, Brazil: comparison between percutaneous and intradermal testing / [Sensibilização a polens em cães no Paraná, Brasil: comparação entre o teste percutâneo e o teste intradérmico]

Canine atopic dermatitis is an inflammatory, genetic, pruritic and chronic dermatosis that affects between 10 and 30% of dogs and one of the most important allergens is grass pollen. The objective of this study was to evaluate the sensitization to grass pollen allergens in dogs with canine atopic dermatitis and to compare intradermal skin test (IDT) with percutaneous test (PT). For this study, ten healthy dogs and 39 dogs with atopic dermatitis were tested. Dogs were submitted to IDT and PT for Lolium multiflorum, Cynodon dactylon and Paspalum notatum. The IDT and PT tests were compared using the Proportion Test. All healthy dogs were negative to both tests. Ten atopic dogs (25.6%) responded positively to the PT and none were positive in IDT. C. dactylon, L. multiflorum and P. notatum were responsible for positive reactions in 70%, 70% and 30% of positive dogs, respectively. The number of positive reactions in PT were statistically higher than IDT (P<0.05). In conclusion, grass pollen can be important source of allergens for dogs in Paraná state (Brazil) and the PT showed higher sensitization to grass pollen in dogs with atopic dermatitis than IDT.(AU)

A dermatite atópica canina é uma dermatose inflamatória, genética, prurítica e crônica que afeta entre 10% e 30% dos cães, e um dos alérgenos mais importantes são os polens de gramíneas. O objetivo deste estudo é avaliar a sensibilização a alérgenos de polens de gramíneas em cães com dermatite atópica e comparar o teste intradérmico (TID) com o teste percutâneo (TP). Para o estudo, 10 cães hígidos e 39 cães com dermatite atópica foram testados. Estes foram submetidos ao TID e ao TP para Lolium multiflorum, Cynodon dactylon e Paspalum notatum. TID e TP foram comparados usando-se o teste de proporção. Todos os cães hígidos foram negativos em ambos os testes. Dez cães atópicos (25,6%) responderam positivamente ao TP e nenhum ao TID. C. dactylon, L. multiflorum e P. notatum foram responsáveis por reações positivas de 70%, 70% e 30% dos cães positivos, respectivamente. O número de reações positivas no TP foi estatisticamente maior que no TID (P<0,05). Foi concluído que os polens de gramíneas podem ser importantes fontes de alérgenos para cães no estado do Paraná (Brasil) e que o TP mostrou maior sensibilização a polens em cães com dermatite atópica que o TID.(AU)

Genetic analysis of a possible case of canine X-linked ectodermal dysplasia.

In the present report, we describe targeted next-generation sequencing of the EDA gene of a male poodle with a clinical and histopathological diagnosis of X-linked hypohidrotic ectodermal dysplasia. The result was compared with the reference sequence and with the result of the sequencing of a normal dog's EDA gene. No point variant, small deletion or insertion were found in the exons and splice sites, but a transition and a transversion were found in the intron 6' and 3' UTR, respectively. The cause of the dysplasia of the affected dog in this study is neither a point variant nor a small deletion or insertion in the exons and splice sites of the EDA gene. Therefore, patients with phenotype of XLHED may have other types of variants in the EDA gene or variants in other genes of the EDA signalling pathway.

Epitheliotropic lymphoma with heart and kidney metastasis in a dog: case report / Linfoma epiteliotrópico em cão com metástase cardíaca e renal: relato de caso

A female adult dog, with a four-month history of pain and intense pruritus, which eventually resulted in sudden death, was referred for necropsy. Postmortem examination showed thoracic and abdominal serum-sanguineous exudates, multifocal infiltrative renal masses, and similar tumors in the heart. Histopathology revealed midsize infiltrative neoplastic proliferation composed of round cells, sparse cytoplasm, and large hyperchromatic nuclei. Immunohistochemistry revealed CD3+ and CD20-immunoexpression. Histopathological and immunohistochemical findings confirmed the diagnosis of epitheliotropic lymphoma with cardiac and renal metastasis.(AU)

Foi encaminhado para necropsia um cão adulto do sexo feminino, com histórico de dor e prurido intenso com evolução de quatro meses, que acabou resultando em morte súbita. O exame post mortem mostrou presença discreta de exsudato serossanguinolento em cavidades torácica e abdominal, massas renais infiltrativas multifocais e tumores semelhantes no coração. O exame histopatológico revelou proliferação neoplásica infiltrativa composta de células redondas, com citoplasma escasso, e grandes núcleos hipercromáticos. A análise imuno-histoquímica mostrou imunoexpressão CD3+e CD20. Os achados histopatológicos e imuno-histoquímico confirmaram o diagnóstico de linfoma epiteliotrópico com metástase cardíaca e renal.(AU)

Comparative study of histopathology and immunohistochemistry of indefinite round cell cutaneous tumors and characterization of canine lymphoma / Estudo comparativo de histopatologia e imuno-histoquímica de tumores indefinidos de células redondas e caracterização de linfoma canino

With the purpose of shedding light on some doubts in veterinary oncology, the present article intends to compare the results of histopathological and immunohistochemical examinations of unspecific round cell neoplasia, to realize immunophenotyping of canine lymphoma cases, to establish the T or B origin of neoplastic cells, and to determine the degree of proliferation and apoptosis of lymphomas by immunohistochemistry. Of 11 animals presenting immunohistochemical diagnosis of lymphoma, five had been diagnosed as Lymphoma by HE staining of histopathological slides and six had been classified as unspecific round cell neoplasia. All cases submitted to immunohistochemical examination were T-cell lymphomas. There was a positive correlation between cell proliferation and apoptosis. The comparison among histopathological and immunohistochemical results obtained in the cases examined in the present study suggested that immunohistochemistry is essential for the differentiation of round cell neoplasia.

Com o objetivo de sanar algumas dúvidas na área da oncologia veterinária, o presente artigo pretende comparar os resultados dos exames histopatológicos e imuno-histoquímicos de neoplasias de células redondas inespecíficas, realizar imunofenotipagem dos casos de linfoma canino e determinar o grau de proliferação e apoptose de linfomas. Dos 11 animais que apresentaram diagnóstico imuno-histoquímico de linfoma, cinco foram diagnosticados como linfoma por coloração HE das lâminas histopatológicas e seis foram classificados como neoplasia de células redondas inespecíficas. Todos os casos submetidos ao exame imuno-histoquímico foram de linfomas de células-T. Houve uma correlação positiva entre a proliferação celular e apoptose. A comparação entre os resultados histopatológicos e imuno-histoquímicos obtidos nos casos analisados sugeriu que a imuno-histoquímica é essencial para a diferenciação das neoplasias de células redondas.

Association of subgingival colonization of Candida albicans and other yeasts with severity of chronic periodontitis.

BACKGROUND AND OBJECTIVE: The aim of this study is to analyze the relationship between the subgingival colonization by Candida albicans and other yeasts with the severity of chronic periodontitis (CP). MATERIAL AND METHODS: After sample size calculation, 40 patients with CP and 20 healthy subjects (HS) were included in the study. Cases of slight-moderate (MCP, n = 23) and severe CP (SCP, n = 17) were defined according to the Centers for Disease Control/American Association of Periodontology classification. Subgingival samples were acquired using sterile paper-points from the sulcus or the deepest periodontal pocket of each healthy and subject with CP, respectively, and were cultured aerobically on three selective media. Yeast colonies that grew on the surface of plates were later identified by biochemical reactions. Statistical tests were used to analyze the association between subgingival yeast colonization (number of yeast-positive individuals and colony forming units (CFU) per subject) and periodontal disease status, considering statistical significance when P < 0.05. RESULTS: Although several yeast species were found (C. parapisilosis, Rhodotorula sp., C. dubliniensis and C. tropicalis), only C. albicans was present in all the patients with yeast-positive CP. Twelve patients (30%) with CP presented yeasts in the subgingival biofilm while only three patients (15%) in the HS group were positive for these microorganisms. No statistical difference was found between the CP and HS groups (P = 0.084). However, when the CP group was divided on the basis of severity, statistical differences were observed between the SCP and MCP groups (47% vs. 17%, P = 0.043), and between the SCP and HS groups (47% vs. 15%, P = 0.033). No statistical difference was observed between the MCP and HS groups (17% vs. 15%, P = 0.832). High densities of yeasts were found only in patients with MCP and SCP (mean and range 61.25 (0-100) CFU/plate and mean and range 51 (0-101) CFU/plate, respectively). CONCLUSION: In this group of patients, subgingival colonization of some yeasts, especially C. albicans, was associated with the severity of CP.

Antiviral activity-guided fractionation from Araucaria angustifolia leaves extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Araucaria angustifolia (Bert.) O. Kuntze (Araucariaceae) is a Brazilian medicinal plant traditionally used for the treatment of various illnesses including dried skin, wounds, shingles, and sexually transmitted diseases. AIM OF THE STUDY: The rationale of the study was to provide evidence of its antiherpes activity in order to confirm its popular use that could be related to herpes disease. MATERIALS AND METHODS: The crude hydroethanolic extract (HE) obtained from Araucaria angustifolia leaves was submitted to a sequential liquid-liquid extraction with solvents of increased polarity. The HE and fractions obtained were evaluated for cytotoxicity and antiherpes activity (Herpes Simplex Virus type 1) by MTT assay. The most active fractions were selected to perform an in vitro antiviral activity-guided chromatographic fractionation. RESULTS: The ethyl acetate (EA) and n-butanol (NB) fractions have shown the best results for antiherpetic activity and their further fractionation yielded 22 subfractions. From these subfractions, 14 were active, and the most potent antiherpetic activity was obtained for NB1-4 subfraction with selectivity index (SI) of 57.51. Chemical analysis of NB1-4 subfractions revealed the presence of proanthocyanidins and the known biflavonoids (bilobetin, II-7-O-methyl-robustaflavone and cupressuflavone). The same biflavonoids have been detected in EA subfractions. CONCLUSION: The present study has shown that the hydroethanolic extract from Araucaria angustifolia leaves as well as many different fractions and subfractions exhibited antiherpes activity, supporting the use of this plant species in folk medicine.

Síndrome de Sézary em cadela / Sézary syndrome in a bitch

The present report describes a case of Sezary syndrome in a canine with lymphadenomegaly, generalized erithroderma, intense pruritus and disseminated cutaneous nodules and plaques. Biopsy samples were taken from cutaneous nodules and plaques and were diagnosed epitheliotropic T cell cutaneous lymphoma by histology and immunohistochemical stain. Bone marrow cytology confirms leukemia. Diagnosis of Sezary syndrome was achieved through clinical, hematological, citopathological, histopathological and immunohistochemical findings. The patient was treated with Madison-Wisconsin chemotherapy protocol, but died after two mouths of treatment

Cutaneous pyogranuloma in a cat caused by virulent Rhodococcus equi containing an 87 kb type I plasmid.

A 2-year-old intact male domestic shorthaired cat presented with a chronic, nodular, ulcerated, cutaneous lesion on the right thoracic limb. Histological and cytological examination revealed a pyogranulomatous inflammation with basophilic organisms in the macrophages. A virulent form of Rhodococcus equi containing an 87 kb type I (VapA) virulence plasmid was identified from cultures of biopsy samples. This report describes the clinicopathological features, plasmid profile and virulence of this case of R equi infection.

Comparação das metodologias da Farmacopéia Brasileira para determinação de água em matérias-primas vegetais, e validação da determinação de água em analisador de umidade para Calendula officinalis L., Foeniculum vulgare Miller, Maytenus ilicifolia Mart. ex. Reissek e Passiflora alata Curtis / Comparison of pharmacopeial methods for water determination in plant raw materials and validation of water determination by infrared drying for Calendula officinalis, Foeniculum vulgare, Maytenus ilicifolia e Passiflora alata

No presente trabalho foi descrita a comparação da aplicabilidade das metodologias preconizadas na Farmacopéia Brasileira (1988) para a determinação de água em drogas vegetais. Foi realizada, ainda, a validação da determinação de água em analisador de umidade. As matérias-primas vegetais analisadas foram Calendula officinalis L. (calêndula), Foeniculum vulgare Miller (funcho), Maytenus ilicifolia Mart. ex. Reissek (espinheira-santa) e Passiflora alata Curtis (maracujá). A análise dos resultados obtidos mostrou que há diferenças estatisticamente significativas (p < 0,05) entre as metodologias "Determinação de água por destilação azeotrópica" e "Determinação de água em drogas vegetais" em estufa, assim como, entre a "Determinação da perda por dessecação" para as drogas vegetais calêndula e funcho. No entanto, não foram observadas diferenças estatisticamente significativas (p < 0,05) entre as duas metodologias de secagem em estufa. Quanto à validação da determinação de água em analisador de umidade, não foram observadas diferenças estatisticamente significativas (p<0,05) entre os resultados das análises em analisador de umidade e os obtidos através da "Determinação da perda por dessecação". Além disso, não foram verificadas diferenças estatisticamente significativas (p < 0,05) para as análises referentes à repetibilidade, precisão intermediária e reprodutibilidade da técnica, o que comprova a exatidão e a precisão da metodologia proposta.

A comparison of the Brazilian pharmacopeial methodologies for water determination in plant raw materials is presented for the following species: Calendula officinalis, Foeniculum vulgare, Maytenus ilicifolia e Passiflora alata. In addition, the validation of water determination by infrared drying was carried out for these species. The results obtained using the azeotropic distillation method (Brazilian Pharmacopoeia - "Determination of Water by Azeotropic Distillation") were significantly different (p<0.05) than those obtained using two different gravimetric methods (Brazilian Pharmacopoeia - "Determination of Water in Vegetable Drugs" and "Determination of loss on Drying"). However, no significant statistical difference was observed between the two gravimetric methodologies. After optimization of operating conditions, the results of water determination by infrared drying were not significantly different than those obtained using the loss on drying pharmaceutical method. Also, no statistically significant differences were observed for repeatability, intermediate precision and reproducibility of the method, demonstrating the accuracy and precision of the proposed methodology.

Antiviral evaluation of plants from Brazilian Atlantic Tropical Forest.

The antiviral activity of six medicinal plants from Brazilian Atlantic Tropical Forest was investigated against two viruses: herpes simplex virus type 1 (HSV-1) and poliovirus type 2 (PV-2). Cuphea carthagenensis and Tillandsia usneoides extracts showed the best antiherpes activity. T. usneoides dichloromethane, ethyl acetate and n-butanol extracts, and Lippia alba n-butanol extract showed inhibition of HSV-1, strain 29R/acyclovir resistant. In addition, only L. alba ethyl acetate extract showed antipoliovirus activity. These results corroborate that medicinal plants can be a rich source of potential antiviral compounds.

Demodectic mange in fattening pigs in São Paulo, Brazil.

An investigation was conducted of mange in five Large White adult sows refractory to pyrethroid treatment (cypermethrin 15%). The most important clinical signs consisted of erythema, hyperkeratosis, alopecic macules, micaceous scaling, follicular pluging, and numerous comedones, especially on the facial region. Skin samples were scraped and punched. Elongate mites were found that were classified as Demodex sp., according to the morphological and histopathological characteristics. The protein, vitamin, and mineral levels contained in the rations were considered to be lower than those recommended to attend the requirements of fattening pigs. Adjustment of the rations was adopted, and the treatment of animals with ivermectin premix (450g/tonne of food) daily orally for a further 7 days was prescribed. The success of treatment was observed 15 days after adoption of these measures, when the beginning of remission of signs was observed. No mites were found on scrapings or biopsy punches after the treatment of the sows.

Comparative study of radical scavenger activities of crude extract and fractions from Cuphea carthagenensis leaves.

This study investigated the superoxide anion and hydroxyl radical scavenger properties, as well as the inhibition of lipid peroxidation by the crude hydroalcoholic extract (CE) and the butanolic (BF) and ethyl acetate (EAF) fractions of Cuphea carthagenensis leaves. In a enzymatic system of O2- production (xanthine/xanthine oxidase system) the CE, EAF and BF (0.1-100 microg ml(-1)) were effective at inhibiting both uric acid formation and NBT reduction by O2(-1). In the non-enzymatic system of O2- generation, the CE and fractions were effective only at the concentration of 100 microg ml(-1). The CE, EAF and BF were also evaluated for their ability to scavenge hydroxyl radicals and/or to chelate iron. The results showed that CE, BF and EAF from C. carthagenensis (0.1-100 microg ml(-1)) were able to inhibit deoxyribose degradation in a concentration-dependent manner. CE was more potent than the fractions. In a hydrophobic system, increasing concentrations of CE, EAF and BF (0.1-100 microg ml(-1)) caused graded inhibition of lipid peroxidation of rat liver homogenate. The EAF displayed the lowest median inhibitory concentration. The present study suggests that an extract (CE) and fractions (EAF and BF) from C. carthagenensis leaves are significant sources of phenolic compounds with antioxidant activity in vitro and may have important health effects, for example, in cardiovascular disease.

Butanolic fraction from Cuphea carthagenensis Jacq McBride relaxes rat thoracic aorta through endothelium-dependent and endothelium-independent mechanisms.

This study evaluated the vasorelaxant properties of the crude hydroalcoholic extract (CE) of Cuphea carthagenensis, as well as its butanolic (BF) and ethyl acetate (EA) fractions, in rings of rat thoracic aorta. In endothelium-intact rings contracted with phenylephrine (30-100 nM), cumulative additions of increasing concentrations of CE, BF, and EA of C. carthagenensis (0.1 microg/ml-3 mg/ml) caused graded relaxations, with BF displaying the lowest median inhibitory concentration (IC5; mean, 6.8 microg/ml; 95% confidence limits, 3.3-14.2). BF-induced relaxations of endothelium-intact rings were virtually abolished by prior incubation with the NO-synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG; 10 or 30 microM), and were markedly reduced after guanylate cyclase inhibition with either methylene blue (10 microM) or ODQ (1 microM; 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). The inhibition of BF-induced relaxation by L-NOARG was prevented to a large extent by simultaneous incubation with L-arginine (1 mM). In endothelium-denuded rings contracted with phenylephrine, CE and BF caused graded relaxations only at doses >100 microg/ml, whereas the NO-donors SNAP (S-nitroso-N-acetyl-penicillamine) and SIN-1 (3-morpholino-sydnonimine) induced full relaxation at 1 microM. BF (100 microg/ml), which caused little relaxation per se of endothelium-denuded rings, potentiated the relaxant effects of SNAP and even more so of SIN-1 (which, unlike SNAP, also releases superoxide anion O2- in addition to NO), in a manner qualitatively similar to that seen with SOD (superoxide dismutase) against SIN-1. These data indicate that the BF of C. carthagenensis induces relaxation of the rat thoracic aorta by two mechanisms: (a) an endothelium-dependent component of action, which clearly depends on the NO/cyclic guanosine monophosphate (cGMP) pathway and can be attributed, at least in part, to free radical-scavenging properties; and (b) an endothelium-independent component of action, which becomes evident at higher doses (> or = 100 microg/ml) and remains to be further characterized. These results suggest that this native South American plant might be beneficial in cardiovascular disease.

Anti-inflammatory effects of the products from Wilbrandia ebracteata on carrageenan-induced pleurisy in mice.

Wilbrandia ebracteata Cogn. (Cucurbitaceae) is commonly known in Brazil as "Taiuia". The roots are employed in folk medicine for the treatment of several diseases, such as rheumatic disease. This study has evaluated the anti-inflammatory action of dicloromethane fraction (F-DCM), purified fraction (PFIII) and Cucurbitacin B extracted from crude extract of W. ebracteata in experimental models in vivo. The F-DCM (0.3 to 10 mg.kg(-1), i.p. or 3 to 30 mg.kg(-1) p.o.) produced significant but not dose-dependent inhibition of the carrageenan-induced cell influx and exsudate leakage in the pleural cavity of mice. The F-DCM 0.01 to 10 mg.kg(-1), i.p. or 0.1 to 10 mg.kg(-1) p.o.) decreased the levels of PGE2 in the exsudate leakage induced by carrageenan in the pleural cavity after 4 h with a calculated ID50 of 0.01 (0.002-0.09, i.p.) and 0.29 (0.05-1.45, p.o.) mg.kg(-1). The PFIII (3 mg.kg(-1), i.p.) inhibited 80% of cell migration (1.50 +/- 0.09 x 10(6) cells/cavity) and exsudate leakage by about 50% (3.09 +/- 0.71 microg/ml) in relation to the control group. Cucurbitacin B (0.1 mg.kg(-1), i.p.), the main compound of PFIII, reduced significantly the levels of PGE2 in the exsudate leakage by 40.7% (10.41 +/- 2.67 ng.ml(-1)). These data show that the active principle(s) present in the F-DCM of W. ebracteata elicited pronounced anti-inflammatory effects when assessed by i.p. or p.o. routes, as well as PFIII. The F-DCM was also able to prevent PGE2 formation in exsudate leakage induced by carrageenan, as well as Cucurbitacin B, its active principle. These results indicate that the anti-inflammatory activity of Wilbrandia ebracteata can be related with the inhibition of the production of PGE2.

Analysis of the role of nitric oxide in the relaxant effect of the crude extract and fractions from Eugenia uniflora in the rat thoracic aorta.

This study has evaluated the possible role played by the L-arginine-nitric oxide pathway in the vasorelaxant action of the hydroalcoholic extract from Eugenia uniflora, and fractions from the extract, in rings of rat thoracic aorta. The addition of an increasing cumulative concentration of hydroalcoholic extract from E. uniflora (1-300 micrograms mL-1) caused a concentration-dependent relaxation response in intact endothelium-thoracic aorta rings pre-contracted with noradrenaline (30-100 nM). The IC50 value, with its respective confidence limit, and the maximum relaxation (Rmax) were 7.02 (4.77-10.00) micrograms mL-1 and 83.94 +/- 3.04%, respectively. The removal of the endothelium completely abolished these responses. The nitric oxide synthase inhibitors N omega-nitro-L-arginine (L-NOARG, 30 microM) and N omega-nitro-L-arginine methyl ester (L-NAME, 30 microM), inhibited the relaxation (Rmax) to -10.43 +/- 7.81% and -3.69 +/- 2.62%, respectively. In addition, L-arginine (1 mM), but not D-arginine (1 mM), completely reversed inhibition by L-NOARG. Methylene blue (30 microM), a soluble guanylate cyclase inhibitor, reduced the relaxation induced by the extract to 14.60 +/- 7.40%. These data indicate that in the rat thoracic aorta the hydroalcoholic extract, and its fractions, from the leaves of E. uniflora have graded and endothelium-dependent vasorelaxant effects.

Anti-inflammatory and analgesic effects of cucurbitacins from Wilbrandia ebracteata.

The anti-inflammatory and antinociceptive actions of the CH2Cl2 extract and semipurified fraction (F-III) from roots of Wilbrandia ebracteata Cogn. have been investigated in rats and mice. The CH2Cl2 extract (1-10 mg/kg, i.p.; ID50 5 mg/kg) and (3-30 mg/kg, p.o.; ID50 15 mg/kg) inhibited, in a dose-related manner, carrageenan-induced paw edema in rats. The subfraction (F-III) from CH2Cl2 extract and compounds isolated as cucurbitacin B and E also inhibited carrageenan-induced edema. The CH2Cl2 extract and F-III also exhibited significant analgesic action in acetic acid-induced pain in mice. In the formalin test, the CH2Cl2 extract (0.3-10 mg/kg, i.p.) and (3-30 mg/kg, p.o.) caused inhibition of the neurogenic (first phase) and inflammatory phase (second phase) of formalin-induced pain. However, the CH2Cl2 extract was more effective in relation to the second phase than in inhibition of the formalin-induced edema. These findings suggest that CH2Cl2 extract has potent anti-inflammatory and analgesic action and that F-III and cucurbitacin B and E may account for these actions.

Cucurbitacins as Constituents of Wilbrandia ebracteata.

The methanolic extract from the roots of WILBRANDIA EBRACTEATA afforded, in addition to the known dihydrocucurbitacins B and E, dihydroisocucurbitacin B, Cucurbitacins B, D, E, G, H, P, and 22-deoxycucurbi-tacin D, four new compounds whose structures were elucidated as (20 R,24xi)-3)3beta,16alpha,20)24,25-pentahydroxy-10alpha-cucurbit-5-ene-2,11,22-trione (3- EPI-isocucurbitacin G) ( 8), (20 R,22xi)-2beta16a,20,22,25-pentahydroxy-10alpha-cucurbita-5,23 T-diene-3,11-dione ( 13), (20 R)-25-acetoxy-3,16alpha,20-trihydroxy-30- NOR-2-(beta- D-glucopyranosyloxy)-1,2,3,4,5,10-dehydrocucurbit-6-ene-11,22-dione ( 14), and dihydroisocucurbitacin B-3-glucoside ( 16).