Browsing by Subject "Pharmaceutical biology"

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  • Tukiainen, Kristiina (Helsingfors universitet, 2019)
    Anisakiasis is a parasitic disease caused by larval nematodes of the genus Anisakis. Humans become infected by consuming contaminated raw or undercooked seafood products. Most human infections are caused by Anisakis simplex (A. simplex) complex. Currently there is no effective drug for this global emerging disease. Novel active compounds against the nematode are needed for drug development purposes. The research with A. simplex requires the isolation of the larvae from fish, which is time-consuming, unecological and uneconomical. Thus, the utilization of the model nematode Caenorhabditis elegans (C. elegans) in the research of A. simplex is considered in this study. Activities of Tea tree, Java citronella and Ho wood essential oils against C. elegans were studied. Aim of the assays was to examine whether C. elegans could be used as a model for A. simplex. Observed effects on C. elegans were compared to the previously reported effects on A. simplex. Activity of Tea tree and Java citronella essential oils against A. simplex was also examined to confirm previously reported activity. In addition, activity of six coumarins against A. simplex was investigated. The aim of the assays was to discover novel active compounds against the pathogenic nematode. Four coumarins were tested against C. elegans to examine possible comparable effects. Toxicity studies were performed in aquatic medium in a 6 well plate format (A. simplex) and in a 96 well plate format or in 1.5 mL Eppendorf tubes (C. elegans). Tea tree essential oil showed dose dependent activity against C. elegans, producing 100% mortality with the concentration 20 μL/mL after 24 hours exposure. Compared to A. simplex, two to three times higher doses were required to produce same degree of mortality in C. elegans. By contrast, Java citronella and Ho wood essential oils showed no significant activity against C. elegans. The activity of Tea tree and Java citronella essential oils against A. simplex was confirmed. The tested coumarins displayed no significant activity against the nematodes. Due to the contradictory results, further investigation about the suitability of C. elegans as a model for A. simplex is needed. Differences between the effective concentrations are probably caused by the differences in the biology of the nematodes, which result from the phylogenetic distance. Based on current results, the tested coumarins were excluded as potential antinematodal compounds against A. simplex, due to the lack of any significant activity on this model.
  • Silén, Heidi (Helsingin yliopisto, 2021)
    Antimicrobial resistance is a growing problem worldwide. It has been shown that more than 70% of the bacteria that cause nosocomial infections are resistant to at least one antibiotic commonly used to treat them. Two concomitant phenomena that aggravate the diarrheal disease situation, especially in developing countries, are general contamination (spread of pathogens due to unclean water, poor sanitation, and malnutrition) and resistant bacterial strains (the adverse consequences of infections increase as infections prolong). According to the WHO, foodborne diseases (FBDs) were estimated to have caused approximately 91 million people to become ill and 137,000 deaths in Africa in 2010. The number is about a third of the deaths caused by FBD worldwide. Diarrhea caused about 70% of the FBD burden. Bacteria that cause food poisoning include Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cronobacter sakazakii, Esherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococccus aureus and Yersinia enterocolitica, some of which are discussed in more detail in this master’s thesis; antibiotics against which resistance has developed, how bacteria resist antibiotics, and the emergence of resistance in Africa. The antibiotic resistance of bacteria and the mechanisms of resistance against antimicrobial drugs are also discussed shortly. In addition to food poisoning, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus can cause difficult-to-treat infections such as wounds. In addition, this work has first dealt with antimicrobial plant derived compounds in general and their modes of action, and then focused on compounds, fractions and extracts of species of the genera Combretum, Terminalia, Pteleopsis and Anogeissus, as well as their antibacterial effects and uses in traditional medicine. In addition, the antibacterial mechanisms of action of different groups of compounds have been discussed in more detail. This work also deals with the combination studies of some plant extracts, fractions and compounds with antibiotics. Combination studies with antibiotics have generally been studied less than the antibacterial effects alone or the effects of combinations of many plant extracts, as used in African traditional medicine. The experimental part covers, among other things, the preparation and yield determination of crude extracts (water and methanol) as well as the agar diffusion method, the microdilution method, the Time kill tests and the checkerboard method in interaction tests to determine MIC, MBC and FIC values. Due to the Covid 19 pandemic, study results were obtained only by the agar diffusion method against Bacillus cereus. The most antimicrobial extracts were extracts of species of the genus Terminalia.
  • Silén, Jenna (Helsingin yliopisto, 2021)
    The life cycle of Chlamydia pneumoniae is a biphasic developmental cycle, as a obligate intracellular bacterium, it forms various morphological forms, including elementary bodies, reticulate bodies and aberrant bodies belonging to a persistent form. Due to the bacterial life cycle and the fact that chronication of C. pneumoniae infection and formation of persistent infection as well as pathogenesis is a complex problem involving multiple signaling pathways and affecting several different cells, it is useful to seek medication to influence infection from different stages of the bacterial life cycle. There are several different factors that induce persistence and thus models of persistence. Although the detection of aberrant RBs and thus aberrant bodies in C. pneumoniae infected tissues does not provide complete certainty about chronic infection, the bacterium has been linked to chronic health problems such as atherosclerotic cardiovascular disease and asthma. The aim of the study was to develop a persistence model induced by beta-lactam antibiotics, amoxicillin and penicillin G, in A549 cells by monitoring the size, shape, and number of inclusions using the IPA method and the immunofluorescence staining method for infection. In addition, the antibiotic sensitizing effect of three compounds on pulmonary chlamydial infection was studied. This effect was monitored by examining the recovery of persistent infection and by monitoring the protective effect of the compounds on beta-lactam-induced persistence. The work succeeded in finding an infection model that is well suited for studying beta-lactam persistence. Due to treatment recommendations, pulmonary chlamydial infections are practically treated with beta-lactam antibiotics. Based on the methods used, it was found that amoxicillin concentrations of 10 and 25 µg/ml and penicillin G concentrations of 100 U/ml and 250 U/ml were sufficiently effective to transfer bacteria to a state of persistence. It was found that the amoxicillin persistence model is reversible based on the increase in the size of the inclusions, especially at 25 µg/ml and quantitatively at 10 µg/ml. It was concluded that amoxicillin at a concentration of 10 µg/ml is sufficient to induce persistence in a beta-lactam antibiotic-induced persistence model. Further quantitative studies on the persistence model are needed, such as quantitative PCR based on the OmpA gene to determine more accurate dose-response relationships. Glutathione levels should also be monitored in the persistence model.
  • Rosqvist, Linn (Helsingin yliopisto, 2021)
    Marine invertebrates are a good and relatively unexplored source of bioactive compounds. These bioactive secondary metabolites can have unique structures and mechanisms of actions, since they are produced by organisms, which means their structures are not limited by the fantasy of chemists. Therefore, bioactive secondary metabolites isolated from marine invertebrates are attractive for drug development. Still, there are challenges regarding bioprospecting marine invertebrates. For example, the amount of material is limited and the environment as well as the biodiversity has to be taken into account when gathering the organisms. The aim of this thesis was to perform the first steps of bioprospecting marine invertebrates; extraction, fractionisation, analysis of bioactivity and identification of bioactive metabolites. The samples used in the experiment, gathered from three different locations, were of the sponge Caulophacus arcticus. The goal was not only to identify one or more bioactive metabolites for eventual further analysis, but also to compare the bioactivity of the samples gathered from different locations. The fractionisation was performed using flash fractionisation, which resulted in eight fractions of each extract. These fractions were tested for anticancer, antibacterial and biofilm inhibiting properties. The bioactivity of the fractions was analysed by performing cell viability assays (MTS assays) on four cell lines, antibacterial growth inhibition assays on five strains of bacteria and biofilm inhibition assays on biofilm of S. epidermidis. The active fractions, the fraction right before and after them and the corresponding fractions of the two other samples were further analysed using UHPLC-HR-MS, in order to identify eventually bioactive compounds and determine the elementary composition of these compounds. The most interesting fractions, from which one or more bioactive compounds were to be identified first, were prioritised based on the bioactivity assays. One compound, which was identified as potentially bioactive with a potentially novel elementary composition, was chosen as a target compound for further analysis. Based on the results, it was also possible to draw the conclusion that there were variations as well as similarities in the bioactivity of samples gathered from different locations. Still, further research is needed to determine if the bioactivity of the same fractions from different samples was caused by the same compounds or not. Even if there are challenges regarding bioprospecting of marine invertebrates, it is still useful to keep studying them in order to find new, bioactive compounds. There is a huge need of new drugs, especially for treating cancer and bacterial infections. Therefore, experiments such as this are relevant also in a bigger perspective. The target compound identified in the experimental part of this thesis might be further analysed in order to determine whether it is bioactive and whether it is profitable to develop it further.
  • Reijonen, Visa-Aleksi (Helsingin yliopisto, 2020)
    Making the treatment of these infections even harder is the fact, that Chlamydia pneumoniae can produce persistent forms of itself, which are immune to antibiotic treatment. When the bacteria sense a stress factor, for example the presence of a β-lactam antibiotic or interferon γ, they start producing these persistent forms called aberrant bodies. When the stress factor is removed, the bacteria can switch back to their replicating form and start infecting the tissues again. It is also known, that C. pneumoniae bacteria will trigger persistence when the bacteria migrate from lung epithelia into monocytes. Interestingly the onset of this mode of persistence does not require any other triggers besides the invasion of the monocyte. These persistence mechanisms enable latent, quiet, and recurring infections. This master’s thesis aimed to study the coculture of lung epithelial (HL cells) and monocytes (THP-1 cells), and by utilising the magnetic separation method presented by Kortesoja et al, to find a positive control compound in the prevention of Chlamydia pneumoniae internalisation into the THP-1 cells for said protocol. In these cocultures the inhibitory effect of different compound groups such as lignans present in Schisandra chinensis plant, MAPK-inhibitors, and β2,2-amino acid derivatives in C. pneumoniae migration from HL cells to THP-1 cells was assessed. Statistic relevance was observed in JNK inhibitor SP600125, MAPKAP-kinase-2 inhibitor SB203580, and ERK1/2 inhibitor FR180204 compounds. These compounds inhibited the internalisation of Chlamydia pneumoniae into THP-1 cells in the cell coculture by 61,05 ± 16,63 % (p = 0,0001), 54,06 ± 16,02 % (p = 0,0002), and 36,76 ± 10,33 % (p = 0,009) respectively. SP600125 and SB 203580 compounds also had an inhibitory effect on the internalisation of C. pneumoniae into the THP-1 cells in a cell monoculture (39,98 ± 18,92 %, p = 0,026 and 37,89 ± 19,47 %, p = 0,035 respectively), whereas FR180204 had no statistical significance, even though it inhibited the internalisation of C. pneumoniae into the THP-1 cells in cell monoculture by 27,53 ± 21,17 %. From the compounds used in the experiments, only MAPK inhibitors had an effect in inhibiting the C. pneumoniae internalisation into the THP-1 cells. The most potent compound in said inhibition was the JNK inhibitor SP600125. JNK pathway has been thought to take part in chlamydial infections but only little research has been done. The results of this master’s thesis’ experiments support the thought of JNK enzyme taking part in chlamydial infections but determining how exactly it affects the infection cycle of C. pneumoniae bacteria still needs further investigation.
  • Virtanen, Sonja (Helsingin yliopisto, 2020)
    Parenteral products are sterile products that are administered as injection, infusion or implantation. Administration of the contaminated parenteral product can cause severe consequences such as sepsis meningitis and even death. Most of the parenteral products used at the hospitals needs to be compounded (e.g. dissolved, diluted) before administration. Whenever possible, compounding should be done in biological safety cabinet using aseptic techniques. According to previous studies errors in aseptic techniques are quite common. Aim of this study was to compare three different environments as compounding area and their effect to the sterility of the compounded parenteral product. Based on the results of this study, changes to the protocols of the hospital could be made. Altogether 220 samples were compounded at two pediatric wards at HUS Helsinki University Hospital. Six volunteers (one pharmacist and five nurses) participated from both wards and each compounded 18 samples in three different environments (patient room, medicine room, biological safety cabinet). The samples were tested for the sterility by membrane filtration within 4 hours or after 24 hours of storage in the refrigerator. The investigator used an observation form to observe the compounding procedures. Environmental monitoring (settle plates) and monitoring of personnel (glove samples) were conducted. Almost all compounded samples (99%, n=213/215) were sterile. There were no significant differences in the contamination rate of the compounded samples between different environments. Five of the collected samples were excluded, because they were contaminated during the sterility test. According to observations, aseptic techniques were well followed. However, disinfection of the septum of the medicine bottle, hand hygiene and cleaning of the compounding area were observed to be deficiently completed. Even though there were lot of variation in the environmental and personnel monitoring the results were quite good. Results from the environmental monitoring were compared to the recommended limits of EU GMP for clean areas. One compounded sample was contaminated with Diezia maris and Corynebacterium mycetoides but the contaminants from the other contaminated sample could not be identified. Aseptic techniques were mainly well followed, however compounding should be done in the biological safety cabinet, since the environmental monitoring results show that the biological safety cabinet was only environment which was within the recommendation limits of the EU GMP for the compounding area of parenteral products. Protocols of the hospital could be changed, since there was no correlation between higher contamination rate of settle plates or compounded samples and not wearing mask and hair cover while compounding in the biological safety cabinet.
  • Lähdeniemi, Veera (Helsingin yliopisto, 2021)
    Drug metabolism is a series of enzyme catalysed processes that modify foreign compounds into a form that is more easily excreted from the body. Compounds can affect the activity of metabolizing enzymes and this may lead to toxic concentrations of a drug that is metabolized via the enzyme. With prodrugs, on the other hand, the drug might not achieve its biologically active form and therefore the treatment will not be effective. Recognizing and preventing metabolic interactions is important already in the early stages of drug discovery and development. Cytochrome P450 (CYP) enzyme inhibition is one of the major reasons for adverse drug-drug interactions (DDIs). The inhibition can be time-dependent (TDI), which means that the potency of inhibition increases over time. TDI may be reversible or irreversible, latter being more severe as new enzymes need to be produced in the body to restore the enzymatic activity. IC50 shift assay is a method that gives information of new compounds potential to cause TDI. IC50 shift assay does not show whether the TDI is reversible or irreversible, however further studies, e.g. dialysis assay, can be conducted to find it out. If the study compound is irreversibly bound to the enzyme, the enzyme activity should not recover in the dialysis. The aim of this master’s thesis was to develop a dialysis method that could determine the reversibility of the TDI observed in the IC50 shift assay. A dialysis method conducted with microsomes is described in earlier literature. Known inhibitors (both time-dependent and direct) for four CYP isoforms were studied in this work: CYP1A2 (furafylline and fluvoxamine), CYP2C9 (tienilic acid and sulphaphenazole), CYP2D6 (paroxetine and quinidine) and CYP3A4 (verapamil, azamulin and ketoconazole). IC50 shift assays were conducted to each inhibitor before the dialysis experiment. The studied compounds behaved in the dialysis assay mostly as assumed based on the literature. The workflow from IC50 shift assay to dialysis assay worked successfully and the IC50 shift data could be utilized when choosing the test concentrations for dialysis assay. Both the IC50 shift assay and dialysis assay were reproducible and the deviations between replicates and separate studies were relatively low. The method still requires some optimizing, but so far, the results are promising. In the future the dialysis method may be part of in vitro CYP inhibition studies at Orion Pharma.
  • Niklander, Johanna (Helsingfors universitet, 2018)
    Tämä tutkimus esittelee kasviperäisen nanokuituselluloosageelin (NFC; GrowDex®) arvioinnin kolmiulotteisena (3U) kasvualustana rintarauhasen organogeneesin mallinnuksessa. Tutkimuksen tavoitteena oli tarkastella kasviperäisen in vitro -kasvualustan aiheuttamaa solusäätelyä normaalissa rinnan epiteelisessä solulinjassa, sekä selvittää rintakudoksen rauhasrakenteiden muodostumisessa keskeisen laminiini 111:n (LAM-111) alustaan lisäyksen mahdollisia hyötyjä viljelmille. Tutkimuksen koeasetelmassa NFC:n edustamaa kasvunicheä arvioitiin ihmisen rintaepiteelistä eristetyllä -ja tyvikalvon proteiinikontaktien säätelystä riippuvaisella MCF 10A -solulinjalla. Solujen in vitro -nicheympäristön verrokkimallinnuksessa hyödynnettiin epiteelisen tyvikalvon proteiiniympäristöä edustavaa proteiinirikasta Matrigel™-2,5U -kasvualustaa. Viljelynäytteistä tehtiin aikapisteittäin valomikroskooppiset -sekä histologiset hematoksyliini – eosiini (HE) morfologian arvioinnit, e-kadheriinin, vimentiinin ja β4-integriinin ilmentymisten vasta-aine-analyysit, sekä β1-integriinin, Bim:in ja c-FLIP-L:n lähetti-RNA:n reaaliaikaiset PCR-analyysit. Analyyseissä keskityttiin tarkastelemaan rintarauhasen epiteelin polarisoitumistapahtumassa havaittavaa solusäätelyä ja proteiinien eritystä. LAM-111 -lisän havaittiin edistävän jossain määrin NFC:ssä viljeltyjen sferoidien sisämorfologian kavitaatiota sekä eritettyjen proteiinien sijoittumista sferoidien pintarakenteisiin Matrigel™ -kontrollinäytteiden kaltaisesti, muttei yksinään riittänyt tuottamaan Matrigel™ :ssä havaittua viljelmien homogeenisyyttä. Kokeen natiivi-NFC:ssä sekä NFC-LAM-111:ssä kasvaneiden sferoidien PCR-analyyseissä havaittiin polarisaatiotapahtumaan liittyvää solusäätelyä viljelmien loppuvaiheessa päivänä 28, poiketen vastaavan PCR profiilin ilmentymisestä Matrigel™ -viljelmissä jo päivänä kolme. NFC -olosuhteissa havaittiin myös Matrigel™ -viljelmistä puuttuvia ylimääräisiä, epiteelisiltä vaikuttavia rakenteita, joiden määritteleminen vaatii lisätutkimuksia. NFC todettiin jäykkyyden suhteen helposti muokattavaksi sekä mahdollisesti kudoksen mekaanisia ominaisuuksia jäljitteleväksi 3U -kasvualustaksi. Tämän kokeen tuloksien perusteella muokkaamatonta NFC:tä voidaan ehdottaa soveltuvaksi kasvualustaksi tyvikalvoproteiinien säätelystä riippumattomille solutyypeille, sekä solutyypeille, jotka kykenevät tuottamaan ympärilleen oman kudostyypillisen proteiiniympäristönsä. Kliiniseen käyttöön kelpuuttavat standardivaatimukset täyttävä NFC vaikuttaa lupaavalta materiaalilta räätälöitävien in vitro -kasvualustojen suunnitteluun, ja mahdollisesti tarjoaa rakenneosiltaan tarkasti määritellyn, xenovapaan, ja proteiinilisillä eri solutyypeille säädettävän in vitro -kasvunichen tulevaisuuden jatkotutkimuksiin.
  • Kilpiö, Tommi (Helsingin yliopisto, 2021)
    Plant cell culture can be used for the production of valuable secondary metabolites. Inspired by the previous studies focusing on capsaicinoid production, this study aimed for establishing plant cell cultures of Capsicum chinense to produce capsinoids. Capsinoids are non-pungent capsaicinoid analogues with potential health benefits. Another aim of this study was to determine the α-solanine content in Capsicum plants and cell cultures to ensure that no toxic amounts are formed during the cell culture. Cell cultures of non-pungent Capsicum chinense cultivars, Trinidad Pimento and Aji Dulce strain 2, were established, and the cultures were fed with intermediates, vanillin and vanillyl alcohol, to enhance the production. In addition, cell cultures of extremely pungent Trinidad Scorpion cultivar were established and they were fed with vanillyl alcohol to study if this would result in formation of capsinoids instead of capsaicinoids. A high-performance liquid chromatography (HPLC) method with UV detection was validated for determining the capsiate contents of the cell culture samples and fruit samples for comparison. To analyze the α-solanine content of the cell culture samples and leaves and flowers of three cultivars belonging to three different Capsicum species, an HPLC-UV method was validated for this purpose as well. Despite validating a sensitive and specific method for capsiate analysis, no detectable amounts of capsiate were detected in any of the cell culture samples. Cell cultures of pungent cultivars did not produce detectable amounts of capsaicinoids either. Results from analyzing the real fruit samples were in accordance with previous literature reports, and Aji Dulce fruits were found to contain higher amounts of capsiate compared to Trinidad Pimento, although having only one indoor grown Aji Dulce fruit analyzed limits the reliability. The analytical method for determining α-solanine content had problems with internal standard and specificity. This method could be used for making rough estimates about the possible α-solanine content. No hazardous amounts were detected in any of the cell culture samples. Only one sample consisting of Aji Dulce young leaves could contain α-solanine slightly above the limits set for commercial potatoes. Results with flowers of Rocoto San Pedro Orange (C. pubescens) and Aji Omnicolor (C. baccatum) were inconclusive and it couldn’t be ruled out that they might contain large amounts of α-solanine. The reason why capsinoids, or even capsaicinoids, were not detected in the cell culture samples remains unsolved, but it could be speculated that capsinoids might degrade in the cell culture environment or that selection of cultivar or cell line is critical. This study gave further proof to the previous assumptions that chili leaves are safe and should not contain notable amounts of α-solanine.
  • Hämäläinen, Sanni (Helsingfors universitet, 2019)
    Kasvavan antibioottiresistenssin vuoksi terveydenhuollossa tarvitaan uusia antibiootteja ja antibioottien apuaineita. Tästä syystä tässä työssä tutkittiin Combretaceae- ja Annonaceae-heimoihin kuuluvien tansanialaisten lääkekasvien antibakteerisia vaikutuksia. Kansanlääketieteessä näitä kasveja on perinteisesti käytetty mm. bakteerien aiheuttamien sairauksien ja oireiden hoitoon, mikä antaa viitteitä siitä, että ne sisältävät antibakteerisia yhdisteitä. Tutkimuksen tarkoituksena oli pyrkiä löytämään raakauutteita ja neste-nesteuutolla saatuja fraktioita, joilla on mahdollisimman hyvät estovaikutukset bakteerien kasvuun. Lisäksi oli tarkoitus selvittää Annonaceae-heimon lajien sisältämiä yhdisteitä, mikä voi osaltaan auttaa löytämään uusia antibiootteja. Antibakteerisia tutkimuksia tehtiin Combretaceae- ja Annonaceae-heimoihin kuuluvien Combretum-, Terminalia-, Friesodielsia- ja Hexalobus-sukujen lajien uutteille ja fraktioille käyttäen agardiffuusio- ja mikrodiluutiomenetelmiä. Yhteensä 45 eri uutteen ja fraktion estovaikutuksia tutkittiin ruokamyrkytyksiä aiheuttavien Bacillus cereus - ja Salmonella enterica -bakteerien kasvuun. Lisäksi tutkittiin Annonaceae-heimoon kuuluvien Friesodielsia obovata - ja Hexalobus monopetalus -lajien uutteiden ja fraktioiden sisältämiä yhdisteitä käyttäen HPLC-DAD - ja UHPLC/Q-TOF-MS -menetelmiä. Tutkimustulosten perusteella useilla Combretaceae- ja Annonaceae -heimoihin kuuluvilla lajeilla on antibakteerisia vaikutuksia grampositiivista B. cereus -bakteeria vastaan, mutta ei niinkään gramnegatiivista S. enterica -bakteeria vastaan. Aiemmissakin tutkimuksissa on usein, ei kuitenkaan aina, saatu parempia estovaikutuksia grampositiivisten kuin gramnegatiivisten bakteerien kasvuun. Tässä tutkimuksessa parhaimmat tulokset (MIC = 156 µg/ml) saatiin mikrodiluutiomenetelmällä B. cereus -bakteerin kasvun estoon Combretum fragrans -lajin lehtien kuumalla Soxhlet-metanoliuutteella ja Friesodielsia obovata -lajin lehtien metanoliuutteen veteen liukenemattomalla fraktiolla. Tarvitaan kuitenkin vielä paljon lisätutkimuksia, varsinkin H. monopetalus - ja F. obovata -lajien kohdalla, jotta voidaan selvittää, voidaanko tutkituista kasviuutteista ja niiden fraktioista eristää mahdollisia uusia antibiootteja tai antibioottien apuaineita ihmisten ja eläinten infektioiden lääkintään. Annonaceae-heimon lajeista löydettiin samantapaisia yhdisteitä kuin on löydetty aiemmissa tutkimuksissa, mutta 6,8-dimetyyli-monohydroksi-pinosembriiniä karakterisoitiin ensimmäistä kertaa F. obovata -lajista. F. obovata -lajin lehdistä tunnistettiin ensimmäistä kertaa (-)-krotepoksidi ja krotepoksidin johdannaisia. Lisäksi H. monopetalus -lajin juuresta karakterisoitiin ensimmäistä kertaa 3-(2',3'-dihydroksi-3'-metyylibutyyli)-5-(3''-metyylikrotonyyli)indolia, 3-(1,3-dihydroksi-3-metyylibut-2-yyli)-6-(2-hydroksi-3-metyyli-3-butenyyli)indolia sekä heksalobiini C:tä ja D:tä.