Furanocoumarins from Ruta chalepensis with Amebicide Activity.

Entamoeba histolytica (protozoan; family Endomoebidae) is the cause of amoebiasis, a disease related to high morbidity and mortality. Nowadays, this illness is considered a significant public health issue in developing countries. In addition, parasite resistance to conventional medicinal treatment has increased in recent years. Traditional medicine around the world represents a valuable source of alternative treatment for many parasite diseases. In a previous paper, we communicated about the antiprotozoal activity in vitro of the methanolic (MeOH) extract of Ruta chalepensis (Rutaceae) against E. histolytica. The plant is extensively employed in Mexican traditional medicine. The following workup of the MeOH extract of R. chalepensis afforded the furocoumarins rutamarin (1) and chalepin (2), which showed high antiprotozoal activity on Entamoeba histolytica trophozoites employing in vitro tests (IC50 values of 6.52 and 28.95 µg/mL, respectively). Therefore, we offer a full scientific report about the bioguided isolation and the amebicide activity of chalepin and rutamarin.

Two Ecdysteroids Isolated from Micropropagated Lychnis flos-cuculi and the Biological Activity of Plant Material.

Genetically uniform plant material, derived from Lychnis flos-cuculi propagated in vitro, was used for the isolation of 20-hydroxyecdysone and polypodine B and subjected to an evaluation of the antifungal and antiamoebic activity. The activity of 80% aqueous methanolic extracts, their fractions, and isolated ecdysteroids were studied against pathogenic Acanthamoeba castellani. Additionally, a Microtox® acute toxicity assay was performed. It was found that an 80% methanolic fraction of root extract exerts the most potent amoebicidal activity at IC50 of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC50 of 0.07 mg/mL. The acute toxicity of 80% fractions at similar concentrations is significantly higher than that of 40% fractions. Crude extracts exhibited moderate antifungal activity, with a minimum inhibitory concentration (MIC) within the range of 1.25-2.5 mg/mL. To the best of our knowledge, the present report is the first to show the biological activity of L. flos-cuculi in terms of the antifungal and antiamoebic activities and acute toxicity. It is also the first isolation of the main ecdysteroids from L. flos-cuculi micropropagated, ecdysteroid-rich plant material.

Evaluation of Oxasqualenoids from the Red Alga Laurencia viridis against Acanthamoeba.

Acanthamoeba genus is a widely distributed and opportunistic parasite with increasing importance worldwide as an emerging pathogen in the past decades. This protozoan has an active trophozoite stage, a cyst stage, and is dormant and very resistant. It can cause Acanthamoeba keratitis, an ocular sight-threatening disease, and granulomatous amoebic encephalitis, a chronic, very fatal brain pathology. In this study, the amoebicidal activity of sixteen Laurencia oxasqualenoid metabolites and semisynthetic derivatives were tested against Acanthamoeba castellanii Neff. The results obtained point out that iubol (3) and dehydrothyrsiferol (1) possess potent activities, with IC50 values of 5.30 and 12.83 µM, respectively. The hydroxylated congeners thyrsiferol (2) and 22-hydroxydehydrothyrsiferol (4), active in the same value range at IC50 13.97 and 17.00 µM, are not toxic against murine macrophages; thus, they are solid candidates for the development of new amoebicidal therapies.

Amoebicidal and Amoebistatic Effects of Artemisia argyi Methanolic Extracts on Acanthamoeba castellanii Trophozoites and Cysts.

PURPOSE : The present study aimed to investigate the amoebicidal and amoebistatic efects of Artemisia argyi leaf methanolic extract by testing the effects on trophozoites and on cysts. We also determined cytotoxic effect, enzymatic and non-enzymatic antioxidant activities, total phenolic, lavonoid and antioxidative contents of A. argyi. METHODS: A. argyi was harvested from various geographic sites in Ordu province in Turkey. The fresh leaves were subjected to methanolic extraction. In 100 µl culture, different concentrations of A. argyi methanolic extract (in quantities from 1.2, 2.3, 4.7, 9.4, 18.7, 37.4, 74.8 mg/ml) and the same volume of trophozoite/cyst suspension were mixed for the determination of the amoebicidal activity of the plant extract. Human bronchial epithelial cells were treated with the same concentrations of Artemisia extracts to determine cytotoxic potential. RESULTS: Total phenolic and lavonoid contents of the extract were calculated as 261 mg gallic acid/g dry extract and 29 mg quercetin/g dry extract, respectively. Total antioxidant activity was also calculated as 367 mg ascorbic acid/g dry extract. The growth of trophozoites stopped in A. argyi methanolic extract with 50% inhibitory concentrations (IC50)/8 h for 37.4 mg/ ml and 74.8 mg/ml extract solution and had stronger amoebicidal activity on the cysts with IC50/72 h. Artemisia showed stronger inhibitory effects on bronchial epithelial cells at the concentrations of 9.4, 18.7, 37.4 and 74.8 mg/ml. CONCLUSION: The study indicated that A. argyi leaf extract has cytotoxic and anti-amoebic activities.

Correlation of radical-scavenging capacity and amoebicidal activity of Matricaria recutita L. (Asteraceae).

Some Acanthamoeba strains are able to cause Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis (AK) worldwide because of their pathogenicity. The treatment of Acanthamoeba infections is complicated due to the existence of a highly resistant cyst stage in their life cycle. Therefore, the elucidation of novel sources of anti-Acanthamoeba agents is an urgent need. In the present study, an evaluation of the antioxidant and anti-Acanthamoeba activity of compounds in flower extracts of Tunisian chamomile (Matricaria recutita L.) was carried out. Chamomile methanol extract was the most active showing an IC50 of 66.235 ± 0.390 µg/ml, low toxicity levels when checked in murine macrophage toxicity model and presented also antioxidant properties. Moreover, a bio-guided fractionation of this extract was developed and led to the identification of a mixture of coumarins as the most active fraction. These results suggest a novel source of anti-Acanthamoeba compounds for the development of novel therapeutic agents against Acanthamoeba infections.

The effect of viroid infection of citrus trees on the amoebicidal activity of 'Maltese half-blood' (Citrus sinensis) against trophozoite stage of Acanthamoeba castellanii Neff.

In order to promote a local Tunisian product, this study was designed to examine, for the first time, the anti-Acanthamoeba activity (Acanthamoeba castellanii Neff) of the essential oils of Tunisian Citrus sinensis peels (Maltese half-blood) and the effect of viroid plant infection on this activity. To do so, three samples of peels' essential oils were studied: from a healthy plant (Control), a plant inoculated with Citrus exocortis viroid (CEVd) and one inoculated with hot stunt cachexia viroid (HSVd). The samples were extracted by hydrodistillation from dried peels and characterized by GC-MS. Limonene was the major component with a percentage ranging from 90.76 to 93.34% for (CEVd) sample and (Control), respectively. Anti-Acanthamoeba activity of the tested oils was determined by the Alamar Blue® assay. Primary results showed a strong potential anti-Acanthamoeba activity with an IC50 ranging from 36.6 to 54.58 µg/ml for (HSVd) and (CEVd) samples, respectively. In terms of the effect of viroid infection, a strong positive correlation was observed between different chemical classes and anti-Acanthamoeba activity.

THE USE OF EXTRACTS FROM PASSIFLORA SPP. IN HELPING THE TREATMENT OF ACANTHAMOEBIASIS.

Chronic progressive diseases of the central nervous system such as granulomatous amoebic encephalitis, amoebic keratitis, amoebic pneumonitis and also skin infections caused by free-living amoebae (Acanhamoeba spp.) are a significant challenge for pharmacotherapy. This is due to the lack of effective treatment because of encystation, which makes the amoebae highly resistant to anti-amoebic drugs. A very inter- esting and promising source of future drugs in this area are plant materials obtained not only from the habitat but also from plant in vitro culture as an alternative source of biomaterials. Alcoholic extracts from leaves of Passiflora incarnata, P. caerulea, P. alata (Passifloraceae) and from callus cultures were evaluated in vito for amoebicidal activity. Phytochemical analysis showed that all extracts contained phenolic compounds including flavonoids? Biological study revealed that all extracts showed amoebostatic and amoebicidal properties in concentrations from 4 to 12 mg/mL. Extracts of P. alata leaf and callus showed the most effective activities (IC5, 4.01 mg/mL, IC,5 7.29 mg/mL, respectively) after 48 h of exposure, which was correlated with the highest concentration of total phenolics and flavonoids in comparison with other extracts.

Methanolic extract of Peganum harmala exhibit potent activity against Acanthamoeba castellanii cysts and its encystment in vitro.

Acanthamoeba castellanii is member of free living amoeba that may cause painful sight-threatening keratitis and life threatening encephalitis which involves central nervous system. Treatments for both infections are problematic because of the amoebic cysts resistance to therapeutic agents. Here we evaluated in vitro strength of methanolic seed extract of Peganum harmala on Acanthamoeba cysts and its encystment mechanism. Our results revealed seed extracts (1 to 30mg/ml) exhibited amoebicidal effects against Acanthamoeba cysts. Furthermore Acanthamoeba encystment was also inhibited in concentration dependent manner with maximum inhibition at 2µg/ml after 48h incubation. In conclusion, we demonstrated for the first time that methanolic extracts exhibit remarkable inhibition of Acanthamoeba cysts and encystment in vitro which could serve a potential new natural agent against Acanthamoeba.

The Amoebicidal Effect of Ergosterol Peroxide Isolated from Pleurotus ostreatus.

Dysentery is an inflammation of the intestine caused by the protozoan parasite Entamoeba histolytica and is a recurrent health problem affecting millions of people worldwide. Because of the magnitude of this disease, finding novel strategies for treatment that does not affect human cells is necessary. Ergosterol peroxide is a sterol particularly known as a major cytotoxic agent with a wide spectrum of biological activities produced by edible and medicinal mushrooms. The aim of this report is to evaluate the amoebicidal activity of ergosterol peroxide (5α, 8α-epidioxy-22E-ergosta-6,22-dien-3ß-ol isolated from 5α, 8α-epidioxy-22E-ergosta-6,22-dien-3ß-ol) (Jacq.) P. Kumm. f. sp. Florida. Our results show that ergosterol peroxide produced a strong cytotoxic effect against amoebic growth. The inhibitory concentration IC50 of ergosterol peroxide was evaluated. The interaction between E. histolytica and ergosterol peroxide in vitro resulted in strong amoebicidal activity (IC50 = 4.23 nM) that may be due to the oxidatory effect on the parasitic membrane. We also tested selective toxicity of ergosterol peroxide using a cell line CCL-241, a human epithelial cell line isolated from normal human fetal intestinal tissue. To the best of our knowledge, this is the first report on the cytotoxicity of ergosterol peroxide against E. histolytica, which uncovers a new biological property of the lipidic compound isolated from Pleurotus ostreatus (Jacq.) P. Kumm. f. sp. Florida.

Abietane diterpenoids from Salvia sclarea transformed roots as growth inhibitors of pathogenic Acanthamoeba spp.

Amoebae from the genus Acanthamoeba are known agents leading to various diseases such as granulomatous amoebic encephalitis (GAE), a chronic progressive disease of the central nervous system, amoebic keratitis (AK), chronic eye infection, amoebic pneumitis (AP), chronic lung infection, and skin infections. It is known that various synthetic anti-Acanthamoeba substances are ineffective. Therefore, other substances, e.g., natural plant compounds, are the focus of biological investigations regarding anti-parasite activity. In this work, the ability of four abietane diterpenoids (ferruginol, salvipisone, aethiopinone, and 1-oxo-aethiopinone) to inhibit Acanthamoeba growth is reported. All investigated compounds were active against Acanthamoeba growing in vitro. Among them, ferruginol demonstrated the highest activity against Acanthamoeba. This compound inhibited Acanthamoeba growth by about 72% in a 3-day exposure period (IC50 17.45 µM), while aethiopinone and 1-oxo-aethiopinone demonstrated this activity at the level of 55-56%. Salvipisone reduced the growth of Acanthamoeba in vitro culture by 39%. For this compound, the value of IC50 was 701.94 µM after 72 h of exposure.

Antiprotozoal activity against Entamoeba histolytica of plants used in northeast Mexican traditional medicine. Bioactive compounds from Lippia graveolens and Ruta chalepensis.

Amoebiasis caused by Entamoeba histolytica is associated with high morbidity and mortality is becoming a major public health problem worldwide, especially in developing countries. Because of the side-effects and the resistance that pathogenic protozoa build against the standard antiparasitic drugs, e.g., metronidazole, much recent attention has been paid to plants used in traditional medicine around the world in order to find new antiprotozoal agents. We collected 32 plants used in Northeast Mexican traditional medicine and the methanolic extracts of these species were screened for antiprotozoal activity against E. histolytica trophozoites using in vitro tests. Only 18 extracts showed a significant inhibiting activity and among them six plant extracts showed more than 80% growth inhibition against E. histolytica at a concentration of 150 µg/mL and the IC50 values of these extracts were determined. Lippia graveolens Kunth and Ruta chalepensis Pers. showed the more significant antiprotozoal activity (91.54% and 90.50% growth inhibition at a concentration of 150 µg/mL with IC50 values of 59.14 and 60.07 µg/mL, respectively). Bioassay-guided fractionation of the methanolic extracts from these two plants afforded carvacrol (1) and chalepensin (2), respectively, as bioactive compounds with antiprotozoal activity.

In vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction.

ETHNOPHARMACOLOGICAL RELEVANCE: Codiaeum variegatum, grown in many varieties, has been widely used as a houseplant based on its brightly decorative foliage. In addition, a variety of this plant has been used for a long time against bloody diarrhea by the local population in Cameroon. AIM OF THE STUDY: In our previous study, the aqueous extract of this plant and an isolated sub-fraction exhibited significant anti-amoebic activity on axenic culture of Entamoeba histolytica. Due to the medicinal value of these extracts, we promptly initiated to investigate their genotoxic and mutagenic potential in order to assure their safe and rationale usage in traditional healthcare system. MATERIAL AND METHODS: Both extracts were incubated with L5178Y mouse lymphoma cells, primary hepatic cells and HepG2 human hepatocellular carcinoma cells and their genotoxicity and mutagenicity were evaluated by quantifying DNA damage and chromosomal aberrations through comet assay, micronucleus assay and mouse lymphoma mutation assay. RESULTS: The aqueous extract of Codiaeum variegatum is not cytotoxic up to 2000 µg/mL while the amoebicidal fraction is significantly cytotoxic (≤40-55%) on L5178Y mouse lymphoma and HepG2 cells at concentrations higher than 500 µg/mL. Besides, no significant DNA damage and induction of micronucleus formation were identified at concentrations up to 2000 µg/mL. Moreover, the mutagenic potential of these extracts after short (4 h) and long term (24 h) treatment, revealed no significant gene mutation induction. CONCLUSION: The aqueous extract of Codiaeum variegatum and the amoebicidal fraction SF9B are neither genotoxic on non-competent or metabolic competent cell lines, nor mutagenic in mouse lymphoma mutation assay and therefore they could be safely used at lower doses for medicinal purpose.

The evaluation of the amebicidal activity of Eryngium planum extracts.

Selected fractions of ethanolic extracts obtained from leaves and roots of Eryngium planum (Apiaceae) were evaluated in vitro for amebicidal activity against Acanthamoeba castellanii. This free-living ameba is the cause of Acanthamoeba keratitis, which is a painful, vision-impairing disease of the eyes, and chronic granulomatous amebic encephalitis. Treatment is very difficult and not always effective because of encystation, which makes the amebae highly resistant to anti-amebic drugs. The search for novel natural amebicidal agents is still of current interest. Fractions of E. planum ethanolic extract from basal leaves: flavonoid fraction (Lf), flavonoid-saponin fraction (Lf-s), saponin fraction (Ls) and phenolic acids fraction (La) and from roots: saponin fraction (Rs) and phenolic acids fraction (Ra) were assayed for antiamebic activity. In the presence of the saponin fractions and phenolic acid fractions (ranging from 1-5 mg/mL), the number of the trofozoites of Acanthamoeba castellanii viable strain 309 decreased during the experimental period (0-72 h). On the other hand, the flavonoid fraction from leaves showed a stimulating activity on the amebae. Almost all fractions (except the flavonoid fraction) showed a time- and dose-dependent amebistatic activity on the trophozoites. Of the fractions tested, the phenolic acid fraction from roots at the concentration of 5 mg/L showed the amebicidal activity on the trophozoites.

In vitro amoebicidal activity of four Peucedanum species on Acanthamoeba castellanii cysts and trophozoites.

Amoebic keratitis is difficult to treat without total efficacy in some patients because of cysts, which is less susceptible than trophozoites to the usual treatments. The aim of this study is to evaluate the in vitro amoebicidal activity of the methanolic extracts of Peucedanum caucasicum, Peucedanum palimbioides, Peucedanum chryseum, and Peucedanum longibracteolatum, which are endemic in Turkish flora except P. caucasicum. Extracts were evaluated for their amoebicidal activities using an inverted light microscope. In the presence of methanolic extracts (ranging from 1.0 to 32.0 mg/ml), numbers of the viable Acanthamoeba castellani trophozoites and cysts were determined during the experimental process (72nd hour). All of the extracts showed a time and dose-dependent amoebicidal action on the trophozoites and cysts. Among the extracts tested, P. longibracteolatum showed the strongest amoebicidal effect on the trophozoites and cysts. In the case of 32 mg/ml concentration of extract, no viable trophozoites or cysts were determined between 24th and 72nd hour. Similar results were obtained from the extract at 16.0 mg/ml concentration against trophozoites. At this concentration value, number of viable cysts was determined as 10.6 ± 2.1 in the 24th hour. In the presence of 8.0 mg/ml extract solution, no viable trophozoites were determined in the 48th hour. At the same concentration, 51% of the cysts were killed by the extract in the 72nd hour. As expected, cysts were found more resistant to the extracts than the trophozoites.

Screening of the in vitro amoebicidal activities of Pastinaca armenea (Fisch. & C.A.Mey.) and Inula oculus-christi (L.) on Acanthamoeba castellanii cysts and trophozoites.

The aim of this study is to evaluate the in vitro amoebicidal activity of the aqueous extracts of Pastinaca armenea and Inula oculus-christi from Turkey. In the presence of aqueous extracts (ranging from 1.0 to 32.0 mg/ml), numbers of the viable Acanthamoeba castellanii trophozoites and cysts were decreased during the experimental process. Both extracts showed a time- and dose-dependent amoebicidal action on the trophozoites and cysts. Among the extracts tested, I. oculus showed the strongest amoebicidal effect on the trophozoites and cysts. In the presence of 32.00 mg/ml extract solution in the media, no viable trophozoites were determined from the time of 24 h. Moreover, in the presence of 16.00 mg/ml I. oculus-christi extract, no viable trophozoites were detected in 72 h of the experiment. Effectiveness of I. oculus-christi extract was found moderate against the cysts. In the presence of 32.00 mg/ml extract, only 25.3% of the total cysts were killed effectively. In the case of P. armenea, 40.3% and 23.0% of the trophozoites and cysts were killed in the presence of 32.00 mg/ml extract at the end of the experimental process (72 h), respectively.

In vitro amoebicidal activities of Teucrium polium and T. chamaedrys on Acanthamoeba castellanii trophozoites and cysts.

Amoebic keratitis is difficult to treat without total efficacy in some patients because of cysts, which are less susceptible than trophozoites to the usual treatments. The aim of this study is to evaluate the in vitro amoebicidal activity of the methanolic extracts of Teucrium polium and Teucrium chamaedrys. In the presence of methanolic extracts (ranging from 1.0 to 32.0 mg/ml), numbers of the viable Acanthamoeba castellani trophozoites and cysts were decreased during the experimental process. Both extracts showed time- and dose-dependent amoebicidal action on the trophozoites and cysts. Among the extracts tested, T. chamaedrys showed the strongest amoebicidal effect on the trophozoites. In the presence of 16 mg/ml or above extract concentrations, no viable trophozoites were observed within 48 h. In the case of T. polium, no viable trophozoites were observed within 48 h at 32 mg/ml concentration. As expected, cysts were found more resistant to the extracts than the trophozoites.

In vitro amoebicidal activity of ethanol extracts of Arachis hypogaea L., Curcuma longa L. and Pancratium maritimum L. on Acanthamoeba castellanii cysts.

Acanthamoeba castellanii causes amoebic keratitis which is a painful sight-threatening disease of the eyes. Its eradication is difficult because the amoebas encyst making it highly resistant to anti-amoebic drugs, but several medicinal plants have proven to be more effective than the usual therapy. This study aimed to evaluate the in vitro amoebicidal activity of ethanol extracts of Arachis hypogaea L. (peanut), Curcuma longa L. (turmeric), and Pancratium maritimum L. (sea daffodil) on A. castellanii cysts. Acanthamoeba were isolated from keratitic patients, cultivated on 1.5% non-nutrient agar, and then incubated with different concentrations of plant extracts which were further evaluated for their cysticidal activity. The results showed that all extracts had significant inhibitory effect on the multiplication of Acanthamoeba cysts as compared to the drug control (chlorhexidine) and non-treated control, and the inhibition was time and dose dependent. The ethanol extract of A. hypogaea had a remarkable cysticidal effect with minimal inhibitory concentration (MIC) of 100 mg/ml in all incubation periods, while the concentrations of 10 and 1 mg/ml were able to completely inhibit growth after 48 and 72 h, respectively. The concentrations 0.1 and 0.01 mg/ml failed to completely inhibit the cyst growth, but showed growth reduction by 64.4-82.6% in all incubation periods. C. longa had a MIC of 1 g and 100 mg/ml after 48 and 72 h, respectively, while the concentrations 10, 1, and 0.1 mg/ml caused growth reduction by 60-90.3% in all incubation periods. P. maritimum had a MIC of 200 mg/ml after 72 h, while the 20-, 2-, 0.2-, and 0.02-mg/ml concentrations showed growth reduction by 34-94.3% in all incubation periods. All extracts seemed to be more effective than chlorhexidine which caused only growth reduction by 55.3-80.2% in all incubation periods and failed to completely inhibit the cyst growth. In conclusion, ethanol extracts of A. hypogaea, C. longa, and P. maritimum could be considered a new natural agent against the Acanthamoeba cyst.

Antibacterial, anthelmintic and anti-amoebic activity in South African medicinal plants.

Hexane, ethanol and water extracts of plants used by South African traditional healers for treating stomach ailments were screened for antibacterial, anthelmintic and anti-amoebic activities. To evaluate antibacterial activity, the disc-diffusion assay was used against several Gram-positive and Gram-negative species. Minimal inhibitory concentration values were determined with a microdilution assay. Ethanolic extracts showed the greatest activity, and Gram-positive bacteria were the most susceptible microorganisms. The free-living nematode Caenorhabditis elegans was used in two different assays to evaluate anthelmintic activity. A microdilution technique was employed to investigate anti-amoebic activity against the enteropathogenic Entamoeba histolytica. These assays were suitable for the screening of a large number of extracts at one time. Several plants exhibited significant activity against these test organisms.

Amoebicidal compounds from medicinal plants.

A series of the natural constituents with amoebicidal activity isolated from several medicinal plants is shown. A list of the medicinal plants potentially active as amoebicide and/or against dysentery also is demonstrated. The present data grouping of the natural compounds and medicinal plants can be an important source of information for the selection of research plant material by the investigators interested in the discovery of new biologically active compounds as amoebicide.

[Growth inhibition of Entamoeba histolytica and E. invadens produced by pomegranate root (Punica granatum L.)]. / Inhibición del crecimiento de Entamoeba histolytica y E. invadens producida por la raíz del granado (Punica granatum L.).

The purpose of the present work has been to evaluate the biological activity of alkaloids and tannins extracted from roots of Punica granatum L. on axenic cultures from Entamoeba histolytica and E. invadens, strains. Initially, the total aqueous extract and later some of its components obtained by thin layer chromatography were tested. These compounds were identified by chromatography as alkaloids or tannins. The density of amebic cultures was determined with a hemocytometer after 48 and 96 hours of incubation, which was calculated by the difference between number of trophozoites obtained at the times chosen and the number of amoebae inoculated. Two milliliters of aqueous extract had higher activity on cultures from E. histolytica than E. invadens strains, producing growth inhibitions of about 100 and 40 per cent respectively. Alkaloid concentrations of 1 mg/ml had no amebicide activity, however tannins at concentrations of 10 micrograms/ml for E. histolytica, and 100 micrograms/ml for E. invadens were sufficient to produce an growth inhibition about 100 per cent. Tannic acid was also tested on the cultures of E. histolytica observing an high inhibitory activity on the growth, this effect was produced at 0.01 mg/ml was similar to that observed with the tannins mixture.