Antiviral activity of terpenes isolated from marine brown seaweeds against herpes simplex virus type 2.

Herpes simplex virus type 2 (HSV-2) is the most common agent of sexually transmitted infections around the world. Currently, no vaccine is available, and acyclovir is the reference compound in treatment HSV-2 infections. However, the emergence of resistant strains has reduced the efficacy in treatment. Several studies have shown marine seaweed biological activities, but there are no studies yet about the activity anti-HSV-2 of two its secundary metabolites, atomaric acid (1) and marine dolastane (2), isolated from Stypopodium zonale and Canistrocarpus cervicornis respectively. Therefore, we evaluated the anti-HSV-2 activity of compounds 1 and 2. Both compounds showed anti-HSV-2 activity with low cytotoxicity and compound 1 inactivated 90% of the viral particles at 50 µM. Both compounds inhibited the penetration and results in silico indicated the compound 1 as possible therapy alternative anti -HSV-2.

Dictyota and Canistrocarpus Brazilian Brown Algae and Their Bioactive Diterpenes-A Review.

Dictyotaceae algae have gained recognition as prolific producers of diterpenes, which are molecules with significant biotechnological potential. These diterpenes hold immense promise as potential active drug components, making the algae a compelling area of study. The present review aims to present the latest advancements in understanding the biopotential of Brazilian Dictyota and Canistrocarpus brown algae, shedding light on the remarkable diversity and the biological and pharmacological potential of the secondary metabolites they produce. A total of 78 articles featuring 26 distinct diterpenes are reported in this review, with their antiviral potential being the mosthighlighted biological activity. Despite considerable research on these algae and their diterpenes, significant knowledge gaps persist. Consequently, the present review is poised to serve as a pivotal resource for researchers who are actively engaged in the pursuit of active diterpenes beyond the immediate purview. Furthermore, it holds the potential to catalyze an increase in research endeavors centered around these algal species within the geographical confines of the Brazilian coastline. Also, it assumes a critical role in directing future scientific explorations toward a better comprehension of these compounds and their ecological implications.

The first use of LC-MS/MS proteomic approach in the brown mussel Perna perna after bacterial challenge: Searching for key proteins on immune response.

The brown mussel Perna perna is a valuable fishing resource, primarily in tropical and subtropical coastal regions. Because of their filter-feeding habits, mussels are directly exposed to bacteria in the water column. Escherichia coli (EC) and Salmonella enterica (SE) inhabit human guts and reach the marine environment through anthropogenic sources, such as sewage. Vibrio parahaemolyticus (VP) is indigenous to coastal ecosystems but can be harmful to shellfish. In this study, we aimed to assess the protein profile of the hepatopancreas of P. perna mussel challenged by introduced - E. coli and S. enterica - and indigenous marine bacteria - V. parahaemolyticus. Bacterial-challenge groups were compared with non-injected (NC) and injected control (IC) - that consisted in mussels not challenged and mussels injected with sterile PBS-NaCl, respectively. Through LC-MS/MS proteomic analysis, 3805 proteins were found in the hepatopancreas of P. perna. From the total, 597 were significantly different among conditions. Mussels injected with VP presented 343 proteins downregulated compared with all the other conditions, suggesting that VP suppresses their immune response. Particularly, 31 altered proteins - upregulated or downregulated - for one or more challenge groups (EC, SE, and VP) compared with controls (NC and IC) are discussed in detail in the paper. For the three tested bacteria, significantly different proteins were found to perform critical roles in immune response at all levels, namely: recognition and signal transduction; transcription; RNA processing; translation and protein processing; secretion; and humoral effectors. This is the first shotgun proteomic study in P. perna mussel, therefore providing an overview of the protein profile of the mussel hepatopancreas, focused on the immune response against bacteria. Hence, it is possible to understand the immune-bacteria relationship at molecular levels better. This knowledge can support the development of strategies and tools to be applied to coastal marine resource management and contribute to the sustainability of coastal systems.

Diterpenes isolated from Canistrocarpus cervicornis with virucidal activity against HIV-1: an in silico evaluation.

HIV is a public health problem, which makes necessary the development of new drugs. Natural products are known for their anti-HIV potential and a good strategy to suggest its mechanism of action is using in silico tools. Herein, diterpenes 1-3 had the binding mode evaluated in the HIV-1 glycoprotein; and properties ADMET in silico performed. In molecular docking important interactions between the hydrophobic cavity, and 1 and 2 were observed. In the molecular dynamics, 1 remained stable covering the entire hydrophobic cavity and performed hydrogen bond during all simulation. ADMET evaluation showed good properties for the diterpenes. Based on these findings, it was possible to suggest the potential from natural products as entry inhibitor and HIV-1 treatment.

How does the brown mussel Perna perna respond to environmental pollution? A review on pollution biomarkers.

The brown mussel Perna perna (Linnaeus, 1758) is a valuable resource for aquaculture in tropical and subtropical coastal regions. It presents desirable characteristics for biomonitoring, including being sessile, widely distributed and abundant, and is a filter-feeder able to accumulate several classes of pollutants (e.g., metals, hydrocarbons, among others). Mussels' biological responses to pollution exposure can be measured as biomarkers, which include alterations ranging from molecular to physiological levels, to estimate the degree of environmental contamination and its effects on biota. This full review compiles two decades (2000-2020) of literature concerning biological effects on P. perna mussel caused by environmental pollutants (i.e., metals, hydrocarbons, and emerging pollutants), considering environmental and farm-based biomonitoring. Biochemical markers related to mussels' oxidative status were efficient for the biomonitoring of metals (i.e., antioxidant enzymes associated with oxidative damage in biomolecules). Genotoxicity and cytotoxicity indicators (i.e., comet, micronucleus, and neutral red assays) provided a depiction of hydrocarbon contamination. The neutral red assay gave a time-concentration cytotoxic response to a wide range of pollutants, including emerging pollutants (e.g., pharmaceuticals and biocides) and hydrocarbons. Perna perna hemocyte parameters provided a useful approach for biocide biomonitoring. This paper summarizes useful biomarkers from molecular to physiological levels in this mussel species used to identify and quantify the degree of coastal pollution. An integrated biomarker analysis may provide a way to overcome possible biomarker variations and assess multi-polluted sites. Nevertheless, it is necessary to investigate biomarker variations according to natural factors (e.g., season and gonad maturation stage) to standardize them for trustworthy biomonitoring.

Palatability and Physical and Chemical Defenses in Five Annelid Polychaetes from Tropical Brazilian Beaches.

AbstractAlthough researchers have examined numerous marine organisms' compensatory characteristics to minimize predation pressure, few have investigated the defensive mechanisms of polychaetes, despite their diversity. Thus, our study's aim was to evaluate the palatability and defensive strategies, both chemical and structural, in the polychaetes Branchiomma luctuosum, Perinereis anderssoni, Phragmatopoma caudata, Eurythoe complanata, and Timarete sp. Another twofold aim was to determine, by comparing our results with the literature, any latitudinal difference in the defensive strategies of E. complanata and to assess defensive mechanisms and palatability in relation to individuals' mobility, overtness, body regionalization, and color. Specimens were collected at Boa Viagem and Itaipu Beaches in Niterói, Rio de Janeiro, Brazil. In assays, a generalist consumer, the hermit crab Calcinus tibicen, was used to evaluate palatability and defenses. In palatability assays of live tissue of the polychaetes, consumers were offered a fresh piece of each polychaete to gauge their acceptance or rejection; in artificial food assays to test extracts of the polychaetes, artificial bait containing extracts of the polychaetes was offered to crabs to identify any chemical defense. Although the extracts of B. luctuosum, E. complanata, P. caudata (i.e., opercular crown), and Timarete sp. were not palatable to the consumers, extracts of P. anderssoni and P. caudata (i.e., body) were. Moreover, the opercular crown of P. caudata and the branchial crown of B. luctuosum showed evidence of structural and chemical defenses. The results corroborate past findings, suggesting no latitudinal variation in the defensive strategies of E. complanata. In general, less motile and more exposed species, aposematic or dark in color, exhibited greater investment in defensive strategies and unpalatability.

In vitro Studies on The Inhibition of Replication of Zika and Chikungunya Viruses by Dolastane Isolated from Seaweed Canistrocarpus cervicornis.

The lack of vaccines and antiviral treatment, along with the increasing number of cases of Zika virus (ZIKV) and Chikungunya virus (CHIKV) infections, emphasize the need for searching for new therapeutic strategies. In this context, the marine brown seaweed Canistrocarpus cervicornis has been proved to hold great antiviral potential. Hence, the aim of this work was to evaluate the anti-ZIKV and anti-CHIKV activity of a marine dolastane isolated from brown seaweed C. cervicornis and its crude extract. Vero cells were used in antiviral assays, submitted to ZIKV and CHIKV, and treated with different concentrations of C. cervicornis extract or dolastane. The crude extract of C. cervicornis showed inhibitory activities for both ZIKV and CHIKV, with EC50 values of 3.3 µg/mL and 3.1 µg/mL, respectively. However, the isolated dolastane showed a more significant and promising inhibitory effect (EC50 = 0.95 µM for ZIKV and 1.3 µM for CHIKV) when compared to both the crude extract and ribavirin, which was used as control. Also, the dolastane showed a very potent virucidal activity against CHIKV and was able to inhibit around 90% of the virus infectivity at 10 µM. For the ZIKV, the effects were somewhat lower, although interesting, at approximately 64% in this same concentration. Further, we observed that both the extract and the dolastane were able to inhibit the replication of ZIKV and CHIKV at different times of addition post-infection, remaining efficient even if added after 8 hours post-infection, but declining soon after. A synergistic effect using sub-doses of the extract and isolates was associated with ribavirin, inhibiting above 80% replication even at the lowest concentrations. Therefore, this work has unveiled the anti-ZIKV and CHIKV potential of C. cervicornis crude extract and an isolated dolastane, which, in turn, can be used as a preventive or therapeutic strategy in the future.

Inhibition by Marine Algae of Chikungunya Virus Isolated From Patients in a Recent Disease Outbreak in Rio de Janeiro.

Chikungunya virus (CHIKV) infection is one of the most challenging re-emergent diseases caused by a virus, and with no specific antiviral treatment it has now become a major public health concern. In this investigation, 25 blood samples were collected from patients with characteristic CHIKV symptoms and submitted to a virus isolation protocol, which detected 3 CHIKV isolates. These samples were evaluated by sequencing for the characterization of the strains and any homology to viruses circulating in Brazil during a recent outbreak. These viruses were used for the development of antiviral assays. Subsequently, the inhibitory effects of seaweed extracts on CHIKV replication were studied. The marine species of algae tested were Bryothamnion triquetrum, Caulerpa racemosa, Laurencia dendroidea, Osmundaria obtusiloba, Ulva fasciata, and Kappaphycus alvarezii, all of which are found in different countries including Brazil. The results revealed high levels of CHIKV inhibition, including extracts of O. obtusiloba with inhibition values of 1.25 µg/mL and a selectivity index of 420. Viral inhibition was dependent on the time of addition of extract of O. obtusiloba to the infected cells, with the optimal inhibition occurring up to 16 h after infection. Neuron evaluations with O. obtusiloba were performed and demonstrated low toxicity, and in infected neurons we observed high inhibitory activity in a dose-dependent manner. These results indicate that the algal extracts may be promising novel candidates for the development of therapeutic agents against CHIKV infections.

A Review of "Polychaeta" Chemicals and their Possible Ecological Role.

Despite the many publications concerning the isolation of substances and the many reviews of marine natural products, some groups of organisms remain poorly studied, including "Polychaeta". In response, this review covers articles published through December 2016 that address marine natural products produced from polychaetes, with a focus on antipredatory strategies, competitors, fouling, and pathogens. A total of 121 compounds were isolated from 1934 to 2016, which includes halogenated aromatics, proteins, amino acids and Lumazine derivatives most notably-with a defensive function were found in the literature, most frequently in the families Sabellidae, Terebellidae, Glyceridae, and Nereididae. The period of highest discovery of natural products in defensive actions for the group was the 2000s. Polychaetes were addressed in 26 revisions of the total 51 articles analyzed and are less reported than other marine invertebrates such as sponges, cnidarians, mollusks, and tunicates. In sum, the present review provides a basis for future research on the marine chemical ecology of polychaetes.

The Brown Alga Stypopodium zonale (Dictyotaceae): A Potential Source of Anti-Leishmania Drugs.

This study evaluated the anti-Leishmania amazonensis activity of a lipophilic extract from the brown alga Stypopodium zonale and atomaric acid, its major compound. Our initial results revealed high inhibitory activity for intracellular amastigotes in a dose-dependent manner and an IC50 of 0.27 µg/mL. Due to its high anti-Leishmania activity and low toxicity toward host cells, we fractionated the lipophilic extract. A major meroditerpene in this extract, atomaric acid, and its methyl ester derivative, which was obtained by a methylation procedure, were identified by nuclear magnetic resonance (NMR) spectroscopy. Both compounds inhibited intracellular amastigotes, with IC50 values of 20.2 µM (9 µg/mL) and 22.9 µM (10 µg/mL), and selectivity indexes of 8.4 µM and 11.5 µM. The leishmanicidal activity of both meroditerpenes was independent of nitric oxide (NO) production, but the generation of reactive oxygen species (ROS) may be at least partially responsible for the amastigote killing. Our results suggest that the lipophilic extract of S. zonale may represent an important source of compounds for the development of anti-Leishmania drugs.

The red seaweed Plocamium brasiliense shows anti-snake venom toxic effects.

BACKGROUND: Snakebite is considered a neglected tropical disease by the World Health Organization. In Brazil, about 70% of the envenomation cases are caused by Bothrops snakes. Its venom may provoke hemorrhage, pain, necrosis, hemolysis, renal or cardiac failure and even death in victims. Since commercial antivenom does not efficiently neutralize the local toxic effects of venoms, natural products have been tested in order to provide alternative or complementary treatment to serum therapy. Therefore, the present study aimed to evaluate the ability of the seaweed Plocamium brasiliense and its active derivatives to neutralize hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic activities of B. jararaca venom. METHODS: Specimens of P. brasiliense were collected in Rio de Janeiro state, Brazil, dried and submitted to oil extraction using four solvents of increasing polarities, n-hexane (HEX), dichloromethane (DCM), ethyl acetate (ETA) and hydroalcoholic solution (HYD). The solvents were evaporated, yielding HEX, DCM, ETA and HYD extracts. Further, all extracts were dissolved in dimethylsulfoxide. In addition, two monoterpenes (8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene and 1,8-dibromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene) and a cholesterol fraction were isolated from the extract of P. brasiliense prepared in hexane. Algal samples were incubated for 30 minutes with B. jararaca venom, and then tested for lethality; hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic effects. RESULTS: Most of the algal extracts inhibited the toxic effects with different potencies. The DCM extract was the most effective, since it inhibited all types of toxic activity. On the other hand, the HYD extract failed to inhibit any effect. Moreover, the isolated products inhibited proteolysis and protected mice from hemorrhage in 30% of the cases, whereas 8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene inhibited 100% and 20% of the hemorrhagic and proteolytic activities, respectively. None of the algal products were toxic to mice. CONCLUSION: Seaweeds may be a promising source of inhibitors against toxic effects caused by B. jararaca envenomation, which may contribute to antivenom treatment.

Effect of diterpenes isolated of the marine alga Canistrocarpus cervicornis against some toxic effects of the venom of the bothrops jararaca snake.

Snake venoms are composed of a complex mixture of active proteins and peptides which induce a wide range of toxic effects. Envenomation by Bothrops jararaca venom results in hemorrhage, edema, pain, tissue necrosis and hemolysis. In this work, the effect of a mixture of two secodolastane diterpenes (linearol/isolinearol), previously isolated from the Brazilian marine brown alga, Canistrocarpus cervicornis, was evaluated against some of the toxic effects induced by B. jararaca venom. The mixture of diterpenes was dissolved in dimethylsulfoxide and incubated with venom for 30 min at room temperature, and then several in vivo (hemorrhage, edema and lethality) and in vitro (hemolysis, plasma clotting and proteolysis) assays were performed. The diterpenes inhibited hemolysis, proteolysis and hemorrhage, but failed to inhibit clotting and edema induced by B. jararaca venom. Moreover, diterpenes partially protected mice from lethality caused by B. jararaca venom. The search for natural inhibitors of B. jararaca venom in C. cervicornis algae is a relevant subject, since seaweeds are a rich and powerful source of active molecules which are as yet but poorly explored. Our results suggest that these diterpenes have the potential to be used against Bothropic envenomation accidents or to improve traditional treatments for snake bites.

The red seaweed Plocamium brasiliense shows anti-snake venom toxic effects

Background Snakebite is considered a neglected tropical disease by the World Health Organization. In Brazil, about 70% of the envenomation cases are caused by Bothrops snakes. Its venom may provoke hemorrhage, pain, necrosis, hemolysis, renal or cardiac failure and even death in victims. Since commercial antivenom does not efficiently neutralize the local toxic effects of venoms, natural products have been tested in order to provide alternative or complementary treatment to serum therapy. Therefore, the present study aimed to evaluate the ability of the seaweed Plocamium brasiliense and its active derivatives to neutralize hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic activities of B. jararaca venom. Methods Specimens of P. brasiliense were collected in Rio de Janeiro state, Brazil, dried and submitted to oil extraction using four solvents of increasing polarities, n-hexane (HEX), dichloromethane (DCM), ethyl acetate (ETA) and hydroalcoholic solution (HYD). The solvents were evaporated, yielding HEX, DCM, ETA and HYD extracts. Further, all extracts were dissolved in dimethylsulfoxide. In addition, two monoterpenes (8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene and 1,8-dibromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene) and a cholesterol fraction were isolated from the extract of P. brasiliense prepared in hexane. Algal samples were incubated for 30 minutes with B. jararaca venom, and then tested for lethality; hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic effects. Results Most of the algal extracts inhibited the toxic effects with different potencies. The DCM extract was the most effective, since it inhibited all types of toxic activity. On the other hand, the HYD extract failed to inhibit any effect. Moreover, the isolated products inhibited proteolysis and protected mice from hemorrhage in 30% of the cases, whereas 8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene inhibited 100% and ...

Semi-synthesis of oxygenated dolabellane diterpenes with highly in vitro anti-HIV-1 activity.

Research on dolabellane diterpenes of brown algae Dictyota spp. has shown that these diterpenoids have strong anti-HIV-1 activity, but there are not data about antiviral activity of dolabellane diterpenes isolated from octocorals, which are antipodes of those isolated from the brown algae. Dolabellanes 13-keto-1(R),11(S)-dolabella-3(E),7(E),12(18)-triene (1) and ß-Araneosene (2) were isolated from the Caribbean octocoral Eunicea laciniata, and both showed low anti-HIV-1 activity and low toxicity. Since it was shown that oxygenated dolabellanes from algae have better anti-HIV-1 activity, in this work some derivatives of the main dolabellane of E. laciniata1 were obtained by epoxidation (3), epoxide opening (4), and allylic oxidation (5). The derivatives showed significant improvement in the anti-HIV-1potency (100-fold), being compounds 3 and 5 the most active ones. Their high antiviral activities, along with their low cytotoxicity, make them promissory antiviral compounds; and it is worth noting that the absolute configuration at the ring junction in the dolabellane skeleton does not seem to be determinant in the antiviral potency of these diterpeneoids.

Antioxidant activity of natural products isolated from red seaweeds.

The present work describes more than 60 natural products from marine red seaweeds (Rhodophyta) and their antioxidant activities. The results indicate that algae belonging to the order Ceramiales, family Rhodomelaceae are the most promising as potential producers of antioxidants. This activity seems to be related to the ability to synthesize polyphenols and their derivatives, as bromophenols.

Dolabelladienols A-C, new diterpenes isolated from Brazilian brown alga Dictyota pfaffii.

The marine brown alga Dictyota pfaffii from Atol das Rocas, in Northeast Brazil is a rich source of dolabellane diterpene, which has the potential to be used in future antiviral drugs by inhibiting reverse transcriptase (RT) of HIV-1. Reexamination of the minor diterpene constituents yielded three new dolabellane diterpenes, (1R*,2E,4R*,7S,10S*,11S*,12R*)10,18-diacetoxy-7-hydroxy-2,8(17)-dolabelladiene (1), (1R*,2E,4R*,7R*,10S*,11S*,12R*)10,18-diacetoxy-7-hydroxy-2,8(17)-dolabelladiene (2), (1R*,2E,4R*,8E,10S*,11S,12R*)10,18-diacetoxy-7-hydroxy-2,8-dolabelladiene (3), termed dolabelladienols A-C (1-3) respectively, in addition to the known dolabellane diterpenes (4-6). The elucidation of the compounds 1-3 was assigned by 1D and 2D NMR, MS, optical rotation and molecular modeling, along with the relative configuration of compound 4 and the absolute configuration of 5 by X-ray diffraction. The potent anti-HIV-1 activities displayed by compounds 1 and 2 (IC50 = 2.9 and 4.1 µM), which were more active than even the known dolabelladienetriol 4, and the low cytotoxic activity against MT-2 lymphocyte tumor cells indicated that these compounds are promising anti-HIV-1 agents.

Antiplatelet and anticoagulant effects of diterpenes isolated from the marine alga, Dictyota menstrualis.

Cardiovascular diseases represent a major cause of disability and death worldwide. Therapeutics are available, but they often have unsatisfactory results and may produce side effects. Alternative treatments based on the use of natural products have been extensively investigated, because of their low toxicity and side effects. Marine organisms are prime candidates for such products, as they are sources of numerous and complex substances with ecological and pharmacological effects. In this work, we investigated, through in vitro experiments, the effects of three diterpenes (pachydictyol A, isopachydictyol A and dichotomanol) from the Brazilian marine alga, Dictyota menstrualis, on platelet aggregation and plasma coagulation. Results showed that dichotomanol inhibited ADP- or collagen-induced aggregation of platelet-rich plasma (PRP), but failed to inhibit washed platelets (WP). In contrast, pachydictyol A and isopachydictyol A failed to inhibit the aggregation of PRP, but inhibited WP aggregation induced by collagen or thrombin. These diterpenes also inhibited coagulation analyzed by the prothrombin time and activated partial thromboplastin time and on commercial fibrinogen. Moreover, diterpenes inhibited the catalytic activity of thrombin. Theoretical studies using the Osiris Property Explorer software showed that diterpenes have low theoretical toxicity profiles and a drug-score similar to commercial anticoagulant drugs. In conclusion, these diterpenes are promising candidates for use in anticoagulant therapy, and this study also highlights the biotechnological potential of oceans and the importance of bioprospecting to develop medicines.

Natural products from marine algae of the genus Osmundaria (Rhodophyceae, Ceramiales).

The present work is a review of the natural products isolated from red marine algae of the genus Osmundaria (including Vidalia), which intends to encompass their occurrence in the species of this genus, the possible synonymies, their geographic distribution, their structural variety and their biological potential as prototypes for the pharmaceutical industry and as active principles of cosmetics. At the end, we provide a table with these natural products and their biological activities.

Inhibitory effects against pasture weeds in Brazilian Amazonia of natural products from the marine brown alga Dictyota menstrualis.

Fractions of the acetone extract and a mixture of two diterpenes from the marine brown alga Dictyota menstrualis were prepared with the aim of identifying potential effects on the germination of seeds and on elongation of the radicle and hypocotyl of the weeds Mimosa pudica and Senna obtusifolia. The bioassay on seed germination was performed in controlled conditions of 25 degreeC temperature and a 12 hour photoperiod, while the one concerning radicle and hypocotyl elongation was performed at the same temperature, though adopting a photoperiod of 24 hours. The results varied according to the receptor species, the fraction utilized, and its concentration. TLC analysis of the fractions and comparison with isolated products indicated that the diterpenes pachydictyol A and isopachydictyol A were the most abundant compounds in fraction HE, whereas the diterpene 6-hydroxy-dichotomano-2, 13-diene-16, 17-dial (3) was the most abundant compound in fractions DC and EA. Analysis of less polar fractions (in n-hexane, dichloromethane and ethyl acetate) revealed values of less than 30% inhibition. On the other hand, the ethanol/water fraction was the most active in all instances. The biological activity of these fractions must be due to the presence of known diterpenes and/or sulfated polysaccharides isolated in earlier studies.