A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus / Atividade anti-helmíntica in vitro de uma protease cisteínica do látex de Ficus benjamina contra Haemonchus contortus

Abstract Haemonchus contortus is a gastrointestinal nematode that is responsible for high mortality rates in ruminant herds. The resistance of nematodes to synthetic anthelmintics is widespread and requires a continuous search for new bioactive molecules, such as proteins. The objective of this study was to evaluate the anthelmintic potential of a protease purified from the latex of Ficus benjamina against H. contortus . Fresh latex was collected from plants via small incisions in the green stems, the rubber was removed by centrifugation, and the latex protein extract (LPE) was obtained. After LPE fractionation with ammonium sulfate and chromatography of the fraction containing the highest proteolytic activity on CM-cellulose, a cysteine protease (FbP) was purified. FbP has a molecular mass of approximately 23.97 kDa, and its proteolytic activity was stable between pH 6.0 and pH 10 and over a broad temperature range, with optimum activity at 60 °C. FbP inhibited both the development and exsheathment of H. contortus larvae, with 50% effective concentrations of 0.26 and 0.79 mg/mL, respectively. We conclude that this cysteine protease from F. benjamina latex with anthelmintic activity against H. contortus could be a promising alternative for the development of products for use in parasite control programmes.

Resumo Haemonchus contortus é um nematoide gastrintestinal, responsável por altas taxas de mortalidade em rebanhos de pequenos ruminantes. A resistência dos nematoides aos anti-helmínticos sintéticos está generalizada e requer uma busca contínua por novos compostos bioativos, como as proteínas. O objetivo deste trabalho foi avaliar o potencial anti-helmíntico da protease purificada do látex de Ficus benjamina contra H. contortus . O látex fresco foi coletado das plantas por pequenas incisões nas hastes verdes e o extrato proteico de látex (EPL) foi obtido. Após o fracionamento do EPL com sulfato de amônio e cromatografia da fração contendo a maior atividade proteolítica da CM-Celulose, uma protease cisteínica (FbP) foi purificada. A FbP tem massa molecular de cerca de 23,97 kDa, a atividade proteolítica foi estável entre pH 6,0 e pH 10 e ao longo de uma ampla faixa de temperatura, com atividade ótima a 60 °C. A FbP inibiu tanto o desenvolvimento quanto o desembainhamento das larvas de H. contortus, com 50% de inibição nas concentrações de 0,26 e 0,79 mg/mL, respectivamente. Concluímos que esta protease cisteínica do látex de F. benjamina, com ação anti-helmíntica contra H. contortus, pode ser uma alternativa promissora para o desenvolvimento de produtos a serem utilizados em programas de controle de parasitos.

TLC separation and antioxidant activity of flavonoids from Carissa bispinosa, Ficus sycomorus, and Grewia bicolar fruits

BACKGROUND: Carissa bispinosa, Ficus sycomorus, and Grewia bicolar are edible fruit plants that grow in the wild. The plants produce yellow-, red-, and purple-colored fruits and thus can be good sources of flavonoids for fighting oxidative reactions in humans, food, and the pharmaceutical industry. The present study aimed at isolating flavonoids from C.bispinosa, F. sycomorus, and G. bicolar fruits and determining their antioxidant activity using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis 3-ethylbenz-thiaz-oline-6-sulfonic acid (ABTS) model radical assays. METHODS: Analytical and preparative thin-layer chromatography was used to isolate flavonoids from the fruits using methanol/chloroform/hexane (7:2:1,v/v/v) as a mobile phase system. The ABTS and DPPH radical scavenging methods were used to test for the antioxidant activity of the samples, using quercetin and catechin as reference standards. RESULTS:Thin-layer chromatographic profiling revealed two different types of flavonoids from each plant.C. bispinosa yielded two flavonoid bands, Rfvalues 0.11 and 0.38;G. bicolaryielded two flavonoid bands,Rfvalues 0.63 and 0.81; andF.sycomorus also yielded two types of flavonoids, Rfvalues 0.094 and 0.81. All the extracted flavonoids exhibited significant antioxidant activity of over 80% at a concentration of 200 mg/L. The order of radical scavenging activity for the 200-mg/Lsamples is G. bicolar Rf(0.81) >C. bispinosa Rf(0.113) >F. sycomorus Rf(0.094) >F. sycomorus Rf(0.047) >C. bispinosa Rf(0.38) >G. bicolar Rf(0.63).G. bicolar(Rf= 0.81) exhibited antioxidant activitythat was superior to that of catechin. CONCLUSION:The present study results show that C. bispinosa,F. sycomorus,and G. bicolar contain different flavonoid types with significant antioxidant activity of over 80% at a concentration of 200 mg/L. Therefore, the fruits can be used as a source of natural antioxidants which can be used as nutraceuticals to promote health, as preservatives to delay peroxidation of foods, and as flavoring for packed foods.

"Pós de doliarina e ferro": um dos remédios importantes da Farmácia Peckolt / "Doliarina and iron powder": an important medicine at Peckolt Pharmacy

Resumo O farmacêutico Theodoro Peckolt é uma das mais importantes figuras da história da química de produtos naturais brasileira. Como outros farmacêuticos do século XIX que atuavam no Brasil, desenvolveu formulações que comercializava em sua farmácia, localizada no Rio de Janeiro, e que tiveram grande prestígio junto à população e à classe médica. O texto apresenta a relação entre a doença identificada inicialmente como opilação e a terapêutica utilizada no século XIX, destacando uma das formulações da Farmácia Peckolt – “Pós de doliarina e ferro”. O produto tem sua origem no látex da espécie Ficus gomelleira(figueira-branca ou gameleira). O artigo tem entre seus objetivos revelar a composição química, feita por métodos modernos de análise do látex deFicus gomelleira.

Abstract The pharmacist Theodoro Peckolt was one of the most important figures in the history of the chemistry of natural Brazilian products. Like other nineteenth-century pharmacists in Brazil, he developed formulations and sold them at his pharmacy in Rio de Janeiro, and these enjoyed great prestige in the eyes both of the public and the medical community. The article discusses the relation between the illness originally called “opilação” (ancylostomiasis, or hookworm) and nineteenth-century treatment. It focuses especially on Peckolt Pharmacy’s “Doliarina and iron powder,” a formulation extracted from the Ficus gomelleira rubber plant. One of the article’s goals is to use modern methods to analyze Ficus gomelleira and identify the chemical composition of the drug.

Effect of prolonged oral administration of aqueous ficus sycomorus stem-bark extract on testicular size of growing albino rat / Efecto de la administración oral prolongada de extracto acuoso de la corteza de ficus sycomorus en el tamaño testicular de rata albina en crecimiento

Decoction of Ficus sycomorus stem bark is used to treat infertility involving low sperm counts, but the reproductive risk associated with its use is uncertain. The aqueous stem-bark extract was orally administered at 200-600 mg/kg for 30 days to post-pubertal growing albino rats. Within the period, the live body weights, scrotal diameters and testicular size parameters were estimated. The mean live body weight, scrotal diameter of the control and treated rats significantly (p<0.05) increased as the animals grew, but the treatment did not significantly (p>0.05) affect the parameters. There were no significant (p>0.05) variations in mean testicular size parameters in treated and control rats, and no dose-dependent nor time-dependent effects occurred. Sections of the testes of the treated animals showed active spermatogenesis in the seminiferous tubules with no apparent histological lesions. It is concluded that the use of the crude extract of the Ficus sycomorus within our dose range portends minimal male reproductive risk and underscores the need to study its pro-spermatic effect.

La decocción de la corteza del tallo de Ficus sycomorus se usa para tratar la infertilidad relacionada al bajo conteo de espermatozoides, pero es incierto el riesgo reproductivo asociado con su uso. El extracto acuoso de la corteza se administró, en ratas albinas, por vía oral, en una dosis de 200-600 mg/kg, durante 30 días post-crecimiento puberal. Durante el período, fueron controlados los siguientes parámetros: peso corporal in vivo, diámetro escrotal y tamaño testicular. El promedio de peso corporal in vivo y diámetro escrotal de las ratas control y tratadas, tuvieron un incremento significativo (p<0,05) en los animales, pero el tratamiento no afectó significativamente (p> 0,05) esos parámetros. No hubo variaciones significativas (p> 0,05) en la media de los parámetros tamaño testicular en las ratas tratadas y control, ni tampoco se produjeron efectos dependientes de la dosis, ni en función del tiempo. Las secciones de los testículos de los animales tratados mostraron una espermatogénesis activa a nivel de los túbulos seminíferos, sin lesiones histológicas aparentes. Se concluye, que el uso del extracto crudo de Ficus sycomorus, dentro de una dosis de 200-600 mg/kg, presenta mínimo riesgo en la reproducción masculina, sin embargo, se hace necesario estudiar su efecto pro-espermático.

Cytotoxic activity of selected West Indian medicinal plants against a human leukaemia cell line / Actividad citotóxica de plantas medicinales de West Indies contra la Línea celular de la leucemia humana

OBJECTIVE: To assess the cytotoxic activities of crude extracts and solvent fractions of Spermacoce verticillata, Ficus pumila and Flemingia strobilifera against a MT-4 human leukaemia cancer cell line. METHODS: Crude extracts of dried leaves of S verticillata, F pumila and F strobilifera were made by exhaustive methanol extraction, fractions were obtained from sequential extraction of the crude extract using solvents of increasing polarity. Dose responses corresponding to cell survival following 72-hour exposure to the extracts were determined using a leukaemia cancer cell line (MT-4). Cell viability was assessed using the MTT[3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay reading absorbances at 570 nm. Comparisons were made with controls and cell survival, in each sample well, was determined based on the ratio ofthe absorbance ofthe sample to the control. RESULTS: Crude extracts of S verticillata, F pumila and F strobilifera displayed cytotoxicity and the IC50 values were 89 µ/ml, 131 µ/ml and 81 µ/ml, respectively. The petroleum ether and chloroform fractions ofthe crude extracts of S verticillata and F strobilifera showed potent cytotoxic activity but the highest cytotoxic activity was found in the chloroform and butanol fractions of F pumila with IC50 values of 23 µg/ml and 26 µg/ml, respectively. CONCLUSION: The crude extracts of S verticillata, F pumila and F strobilifera were shown to be cytotoxic to the leukaemia cell line, MT-4 and IC50 values were determined. Fractionation of the crude extracts by solvent-solvent extraction enabled determination of the active fractions and their IC50 values. We propose that cytotoxic activity may be due to antioxidant compounds previously isolated from these plants.

OBJETIVO: Evaluar las actividades citotóxica de extractos crudos y las fracciones solventes de Spermacoce verticillata, Ficus pumila y Flemingia strobilifera contra una línea celular de la leucemia humana MT4. MÉTODOS: Se obtuvieron extractos crudos de hojas secas de S verticillata, F pumila y F strobilifera mediante extracción exhaustiva con etanol, y se obtuvieron fracciones a partir de la extracción secuencial del extracto crudo mediante solventes de polaridad creciente. Se determinaron las respuestas a las dosis correspondientes a la sobrevivencia de las células luego de 72 horas de exposición a los extractos, usando una línea celular de leucemia (MT-4). La viabilidad celular fue evaluada usando lecturas de absorbancia a partir del ensayo MTT [3-(4, 5-dimetiltiazol-2-il)-2, 5-difenil tetrazolio bromuro] a 570 nm. Se hicieron comparaciones con los controles. La sobrevivencia celular en cada pozo de muestreo, fue determinada a partir de la tasa de absorbancia de la muestra con respecto al control. RESULTADOS: Los extractos crudos de S verticillata, F pumila y F strobilifera mostraron citotoxicidad y los valores IC50 fueron 89 µ/ml, 131 µ/ml y 81 µ/ml, respectivamente. El éter de petróleo y las fracciones de cloroformo de los extractos crudos de S verticillata y F strobilifera mostraron una potente actividad citotóxica, pero la actividad citotóxica más alta fue hallada en las fracciones de cloroformo y butanol de F pumila con valores IC50 de 23 µ/ml y 26 µ/ml, respectivamente. CONCLUSIÓN: Los extractos de S verticillata, F pumila y F strobilifera demostraron ser citotóxicos a la línea celular MT4 y IC50 se determinaron los valores. El fraccionamiento de los extractos crudos extractos mediante extracción solventes-solventes hizo posible la determinación de las fracciones activas y sus valores IC50. Sugerimos que la actividad citotóxica puede deberse a compuestos antioxidantes previamente aislados a partir de estas plantas.

Nutraceutical effects of garlic oil, its nonpolar fraction and a Ficus flavonoid as compared to vitamin E in CCl4 induced liver damage in rats.

Carbon tetrachloride feeding (3.2g/kg/72hr) for one month increased significantly the serum and tissue lipid profile and deranged the enzyme levels viz; alkaline phosphatase, alanine transaminase, aspartate transaminase, glutathionze reductase, HMGCoA reductase, catalase, gluc.6.PDH and malic enzyme in rats. Simultaneously the lipid peroxidation level in liver was also raised. On administration of garlic oil and its major nonpolar fraction (NPFGO) and a flavonoid isolated from the bark of Ficus bengalensis Linn, viz; leucopelargonin derivative respectively to different groups(100mg/kg/day) the deleterious effects of CCl4 were significantly ameliorated. The liver damage by CCl4 was satisfactorily prevented by these samples as effectively as Vit. E (50 mg/kg/day). The results prove that important nutraceuticals (phytonutrients) like bioflavonoids and theols i.e. allylic sulphide rich fractions give protection from toxins like CCl4. The order of beneficial effects of the drugs are Leucopelargonin > NPFGO > Garlic oil and their effects are comparable to that of vitamin E used at a minimal dose.

Potential antifilarial activity of fruit extracts of Ficus racemosa Linn. against Setaria cervi in vitro.

Effect of alcoholic and aqueous extracts of the fruits of F. racemosa Linn., on the spontaneous movements of both the whole worm and nerve muscle preparation of Setaria cervi and on the survival of microfilariae in vitro was studied. Alcoholic as well as aqueous extracts caused inhibition of spontaneous motility of whole worm and nerve muscle preparation of Setaria cervi characterized by increase in amplitude and tone of contractions. Initial stimulatory effect was not observed with aqueous extract on whole worm preparation, while effect of alcoholic extract on whole worm and nerve muscle preparation was characterized by an increase in amplitude and tone of contractions followed by paralysis. The concentrations required to inhibit the movement of the whole worm and nerve muscle preparation for alcoholic extract of fruits of F. racemosa were 250 and 50 microg/ml, respectively, whereas aqueous extract caused inhibition of the whole worm and nerve muscle preparation at 350 and 150 microg/ml, respectively, suggesting a cuticular barrier. Both alcoholic and aqueous extracts caused death of microfilariae in vitro. LC50 and LC90 were 21 and 35 ng/ml, respectively for alcoholic, which were 27 and 42 ng/ml for aqueous extracts.

Mechanism of action of antiatherogenic and related effects of Ficus bengalensis Linn. flavonoids in experimental animals.

One month treatment of alloxan diabetic dogs with a glycoside, viz. leucopelargonin derivative (100 mg/kg/day) isolated from the bark of F. bengalensis decreased fasting blood sugar and glycosylated haemoglobin by 34% and 28% respectively. Body weight was maintained in both the treated groups while the same was decreased significantly by 10% in the control group. In cholesterol diet fed rats, as the atherogenic index and the hepatic bile acid level and the faecal excretion of bile acids and neutral sterols increased, the HMGCoA reductase and lipogenic enzyme activities in liver and lipoprotien lipase activity in heart and adipose tissue and plasma LCAT activity and the incorporation of labelled acetate into free and ester cholesterol in liver decreased significantly. On treatment with the two ficus flavonoids, viz. leucopelargonin and leucocyanin derivatives and another flavonoid quercetin (100 mg/kg/day) the above said effects except on bile acids and sterols and lipogenic enzymes were significantly reversed in the cholesterol fed rats. However in the treated rats the hepatic level of bile acids and the faecal excretion of bile acids and neutral sterols still further increased and the action of lipogenic enzyme glucose 6 phosphate dehydrogenase was still further decreased. These effects of leucopelargonidin and quercetin were better than that of the second. Toxicity studies are required to be carried out to find out if the ficus flavonoids could be used as health promoters as they are hypocholesterolemic and antioxidant in action.