Activity of ethyl acetate extracts of marine-derived fungi against active and hypoxia-induced dormant Mycobacterium (2025)

Journal Pre-proof Antimycobacterial activity and molecular docking of methanolic extracts and compounds of marine fungi from Saldanha and False Bays, South Africa

Antimycobacterial activity and molecular docking of methanolic extracts and compounds of marine fungi from Saldanha and False Bays, South Africa, 2022

The number and diversity of drugs in the tuberculosis (TB) drug development process has increased over the years, yet the attrition rate remains very high, signaling the need for continued research in drug discovery. In this study, crude secondary metabolites from marine fungi associated with ascidians collected from Saldanha and False Bays (South Africa) were investigated for antimycobacterial activity. Isolation of fungi was performed by sectioning thin inner-tissues of ascidians and spreading them over potato dextrose agar (PDA). Solid state fermentation of fungal isolates on PDA was then performed for 28 days to allow production of secondary metabolites. Afterwards, PDA cultures were dried and solid-liquid extraction using methanol was performed to extract fungal metabolites. Profiling of metabolites was performed using untargeted liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS). The broth microdilution method was used to determine antimycobacterial activity against Mycobacterium smegmatis mc2155 and Mycobacterium tuberculosis H37Rv, while in silico flexible docking was performed on selected target proteins from M. tuberculosis. A total of 16 ascidians were sampled and 46 fungi were isolated. Only 32 fungal isolates were sequenced, and their sequences submitted to GenBank to obtain accession numbers. Metabolite profiling of 6 selected fungal extracts resulted in the identification of 65 metabolites. The most interesting extract was that of Clonostachys rogersoniana MGK33 which inhibited Mycobacterium smegmatis mc2155 and Mycobacterium tuberculosis H37Rv growth with minimum inhibitory concentrations (MICs) of 0.125 and 0.2 mg/mL, respectively. These results were in accordance with those from in silico molecular docking studies which showed that bionectin F produced by C. rogersoniana MGK33 is a potential inhibitor of M. tuberculosis β-ketoacyl-acyl carrier protein reductase (MabA, PDB ID = 1UZN), with the docking score observed as -11.17 kcal/mol. These findings provided evidence to conclude that metabolites from marine-derived fungi are potential sources of bioactive metabolites with antimycobacterial activity. Even though in silico studies showed that bionectin F is a potent inhibitor of an essential enzyme, MabA, the results should be validated by performing purification of bionectin F from C. rogersoniana MGK33 and in vitro assays against MabA and whole cells (M. tuberculosis).

Selective Killing Of Dormant Mycobacterium tuberculosis By Marine Natural Products

Antimicrobial agents and chemotherapy, 2017

The dormant phenotype acquired by Mycobacterium tuberculosis (Mtb) during infection poses a major challenge in disease treatment since these bacilli are tolerant to front-line drugs. Therefore, it is imperative to find novel compounds that effectively kill dormant bacteria. By screening 4,400 marine natural product samples against dual-fluorescent Mtb under both replicating and non-replicating conditions we have identified compounds that are selectively active against dormant Mtb This validates our strategy of screening all compounds in both assays as opposed to using the dormancy model as a secondary screen. Bioassay guided deconvolution enabled the identification of unique pharmacophores active in each screening model. To confirm the activity of samples against dormant Mtb we used a luciferase reporter assay and CFU enumeration. The structures of five purified active compounds were defined by NMR and mass spectrometry. We identified two lipid compounds with potent activity towards...

Antimycobacterial and cytotoxic activities of extracts from fungal isolates of Lake Magadi

International Journal of Biological and Chemical Sciences, 2018

In this study, antimycobacterial and cytotoxic activities of ethyl acetate extracts of fungal isolates from Lake Magadi were evaluated. The extracts were tested against Mycobacterium madagascariense (MM) and M. indicus pranii (MIP), and cytotoxicity against brine shrimp (Artemia salina) larvae. Fungal strains were identified using sequence comparison of the Internal Transcribed Spacer (ITS) region. Potent antimycobacterial activities against MM were exhibited by extracts from Volutella colletotrichoides, Helicoon richonis, Penicillium limosum, P. sacculum, Aspergillus parasiticus and A. nomius strains that exhibited minimum inhibition concentration (MIC) in the range of 0.19-12.5 mg/mL. On the other hand, significant antimycobacterial effects against MIP was shown by extracts from V. colletotrichoides, H. richonis, A. parasiticus, Fusarium merismoides, A. silvaticus and A. fumigatus strains in the same MIC range. Notable cytotoxic activities of the extracts were from A. versicolor, A. nomius, P. janthinellum and H. richonis strains with LC 50 values ranging from 46.60-98.12 μg/mL. These results indicate that fungi inhabiting Lake Magadi have the ability to produce bioactive metabolites that could be further explored for potential medicinal agents.

Antimicrobial and Mycochemical Analysis of Crude Extracts of Marine and Terrestrial Fungi

International Journal of Advanced Scientific Research and Management, 2019

In search of new bioactive compounds against human pathogens, marine and terrestrial fungi were isolated from Puducherry coastline and Bengaluru respectively. A comparative study was done on both terrestrial and marine fungi with respect to the mycochemical analysis and antimicrobial activity against Staphylococcus aureus, Salmonella typhi, Bacillus cereus, Escherichia coli and Candida albicans. Morphologically identified selected fungi were cultured for the production of secondary metabolites in Potato Dextrose Broth medium to obtain the crude extract using ethyl acetate. Mycoconstituents revealed the presence of tannins, terpenoids, steroids, phenolics and flavanoids in marine derived fungi SW13, in SW7 only phenolics and tanins were present. Terrestial fungi SS3 showed the presence of phenolics, tanins, flavanoids whereas SS7 showed the absence of all myco-constituents. The marine derived fungi SW13 gave effective inhibition to all the tested pathogens, whereas SW7 gave effective inhibition against S. typhi, B. cereus and C. albicans. While, terrestrial fungi SS3 recorded inhibition against S. aureus, S. typhi and B. cereus. SS7 showed no antimicrobial activity against pathogens.

Activity of Medicinal Plant Extracts on Multiplication ofMycobacterium tuberculosisunder Reduced Oxygen Conditions Using Intracellular and Axenic Assays

International Journal of Microbiology, 2016

Aim.Test the activity of selected medicinal plant extracts on multiplication ofMycobacterium tuberculosisunder reduced oxygen concentration which represents nonreplicating conditions.Material and Methods.Acetone, ethanol and aqueous extracts of the plantsAcorus calamusL. (rhizome),Ocimum sanctumL. (leaf),Piper nigrumL. (seed), andPueraria tuberosaDC. (tuber) were tested onMycobacterium tuberculosisH37Rv intracellularly using an epithelial cell (A549) infection model. The extracts found to be active intracellularly were further studied axenically under reducing oxygen concentrations.Results and Conclusions.Intracellular multiplication was inhibited ≥60% by five of the twelve extracts. Amongst these 5 extracts, in axenic culture,P. nigrum(acetone) was active under aerobic, microaerophilic, and anaerobic conditions indicating presence of multiple components acting at different levels andP. tuberosa(aqueous) showed bactericidal activity under microaerophilic and anaerobic conditions imp...

Antimycobacterial and cytotoxicity activity of synthetic and natural compounds

Química Nova, 2007

Recebido em 13/7/06; aceito em 10/4/07; publicado na web em 5/10/07 Antimycobacterial and cytotoxicity activity of synthetic and natural compounds. Secondary metabolites from Curvularia eragrostidis and Drechslera dematioidea, Clusia sp. floral resin, alkaloids from Pilocarpus alatus, salicylideneanilines, piperidine amides, the amine 1-cinnamylpiperazine and chiral pyridinium salts were assayed on Mycobacterium tuberculosis H37Rv. N-(salicylidene)-2hydroxyaniline was the most effective compound with a minimal inhibitory concentration (MIC) of 8 µmol/L. Dihydrocurvularin was moderately effective with a MIC of 40 μmol/L. Clusia sp. floral resin and a gallocatechin-epigallocatechin mixture showed MIC of 0.02 g/L and 38 µmol/L, respectively. The cytotoxicity was evaluated for N-(salicylidene)-2-hydroxyaniline, curvularin, dihydrocurvularin and Clusia sp. floral resin, and the selectivity indexes were > 125, 0.47, 0.75 and 5, respectively.

The evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from Psoralea corylifolia and Sanguinaria canadensis

Journal of Ethnopharmacology, 2002

Extracts from forty-three plant species were selected on account of reported traditional uses for the treatment of TB and/or leprosy. These were assayed for antimycobacterial activities. A simple in vitro screening assay was employed using two model species of mycobacteria, M. aurum and M. smegmatis. Crude methanolic extracts from three of the plants, C. mukul, P. corylifolia and S. canadensis, were found to have significant antimycobacterial activity against M. aurum only (MIC = 62.5 mg/ml). Bioassay guided fractionation led to the isolation of two known benzophenanthridine alkaloids, sanguinarine (1) and chelerythrine (2), from the roots S. canadensis and the known phenolic meroterpene, bakuchiol (3) from the seeds of P. corylifolia. The fractionation of the resin of C. mukul lead to a decrease in antimycobacterial activity and hence further work was not pursued. Compound (2) was the most active against M. aurum and M. smegmatis (IC 50 = 7.30 mg/ml [19.02 mM] and 29.0 mg/ml [75.56 mM], respectively). M. aurum was the most susceptible organism to all three compounds. No significant difference in antimycobacterial activity was observed when the two alkaloids were tested for activity in media of differing pH values. The activities of the pure compounds against M. aurum were comparable with those against M. bo6is BCG with compound (2) being the most active (M. bo6is BCG, IC 50 = 14.3 mg/ml [37.3 mM]). These results support the use of these plants in traditional medicine.

Identification of Antimycobacterial Natural Products from a Library of Marine Invertebrate Extracts

Medicines, 2022

Tuberculosis (TB) remains a public health crisis, requiring the urgent identification of new anti-mycobacterial drugs. We screened several organic and aqueous marine invertebrate extracts for their in vitro inhibitory activity against the causative organism, Mycobacterium tuberculosis. Here, we report the results obtained for 54 marine invertebrate extracts. The chemical components of two of the extracts were dereplicated, using 1H NMR and HR-LCMS with GNPS molecular networking, and these extracts were further subjected to an activity-guided isolation process to purify the bioactive components. Hyrtios reticulatus yielded heteronemin 1 and Jaspis splendens was found to produce the bengamide class of compounds, of which bengamides P 2 and Q 3 were isolated, while a new derivative, bengamide S 5, was putatively identified and its structure predicted, based on the similarity of its MS/MS fragmentation pattern to those of other bengamides. The isolated bioactive metabolites and semi-pur...

Antimycobacterial evaluation and preliminary phytochemical investigation of selected medicinal plants traditionally used in Mozambique

Journal of …, 2011

Ethnopharmacological relevance: Several medicinal plants are traditionally used in Mozambique to treat tuberculosis and related symptoms. Aims of the study: It was aimed to assess the in vitro antimycobacterial activity of crude extracts from fifteen medicinal plants and to reveal main classes of compounds which may account for the activity of extracts. Methods and materials: The plant materials were sequentially extracted by n-hexane, dichloromethane, ethyl acetate, and 70% ethanol. Decoction of each plant material was also prepared according to traditional use. Broth microdilution method was employed to screen extracts against two mycobacterial species: Mycobacterium smegmatis ATCC 607 and Mycobacterium tuberculosis H37Rv. The extracts with minimum inhibitory concentration(s) (MIC) below 125 g/mL were considered active and further tested against different mycobacterial species and strains, namely Mycobacterium tuberculosis H37Ra, Mycobacterium bovis BCG ATCC 35734, Mycobacterium smegmatis mc 2 155, Mycobacterium avium DSM 44156 and DSM 44157. Cytotoxic effect was evaluated against human macrophages from the monocytic THP-1 cells. Main classes of compounds in these active extracts were proposed from their 1 H NMR spectroscopic characterizations. Results: n-Hexane extracts of Maerua edulis and Securidaca longepedunculata, ethyl acetate extract of Tabernaemontana elegans and dichloromethane extract of Zanthoxylum capense were found to possess considerable activity against Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with MIC 15.6-62.5 g/mL. Tabernaemontana elegans ethyl acetate extract displayed strong activity against Mycobacterium tuberculosis H37Rv (MIC 15.6 g/mL). Except for Tabernaemontana elegans ethyl acetate extract which presented potent cytotoxic effects in THP-1 cells (IC 50 < 4 g/mL), the other three plant extracts showed moderate to none toxicity. Based on 1 H NMR spectroscopic analysis, major components in both Maerua edulis and Securidaca longepedunculata n-hexane extracts were linear chain unsaturated fatty acids. Zanthoxylum capense dichloromethane extract contained more complex constituents (mostly phenolic compounds). In the most potent extract, Tabernaemontana elegans ethyl acetate extract, the prominent compounds were identified as indole alkaloids. Conclusions: The pronounced antimycobacterial activity of the medicinal plants Maerua edulis, Securidaca longepedunculata, Zanthoxylum capense, and Tabernaemontana elegans suggested that they might provide compounds which could be potential anti-TB drug leads.

Antibacterial Activities of Naturally Occurring Compounds against Mycobacterium avium subsp. paratuberculosis

Applied and Environmental Microbiology, 2008

The antibacterial activities of 18 naturally occurring compounds (including essential oils and some of their isolated constituents, apple and green tea polyphenols, and other plant extracts) against three strains of Mycobacterium avium subsp. paratuberculosis (a bovine isolate [NCTC 8578], a raw-milk isolate [806R], and a human isolate [ATCC 43015]) were evaluated using a macrobroth susceptibility testing method. M. avium subsp. paratuberculosis was grown in 4 ml Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase, 0.05% Tween 80 (or 0.2% glycerol), and 2 μg/ml mycobactin J supplemented with five concentrations of each test compound. The changes in the optical densities of the cultures at 600 nm as a measure of CFU were recorded at intervals over an incubation period of 42 days at 37°C. Six of the compounds were found to inhibit the growth of M. avium subsp. paratuberculosis . The most effective compound was trans -cinnamaldehyde, with a MIC of 25.9 μg/ml, foll...

Screening of some New Caledonian and Vanuatu medicinal plants for antimycobacterial activity

Journal of Ethnopharmacology, 2005

Twenty plants, belonging to sixteen families, used in traditional New Caledonian and Vanuatu medicine for treatment of symptoms potentially related to tuberculosis (cough, fever or inflammation) were screened for antimycobacterial activity. We also screened an original endemic plant, Amborella trichopoda, only member of the monogeneric family Amborellaceae and considered the most primitive living angiosperm. In total, 55 extracts were evaluated for inhibitory activity against Mycobacterium bovis BCG strain at a concentration of 100 g/ml. Methanolic and dichloromethane extracts of Amborella trichopoda, Codiaeum peltatum, Myristica fatua, and essential oils Myoporum crassifolium showed an activity at this concentration. Methanolic extract of Amborella trichopoda fruits presented a significant activity with a minimal inhibitory concentration included between 1 and 2.5 g/ml. In the same conditions, this activity was comparable with those of the reference drugs pyrazynamide and ethambutol, at 20 and 2.5 g/ml, respectively.

Effect of ethanomedicinal plants on Mycobacterium smegmatis Tubercular bacilli using mycobacteria growth indicator tube assay

International Journal of Herbal Medicine

Despite all of the control approaches, tuberculosis (TB) is a major cause of death worldwide and onethird of the world's population is infected with TB. Plant-derived medicines have been used in traditional medicinal system for the treatment of many ailments worldwide. From last number of years, plants have advantageous in different type of diseases producing in human beings. The present aim to carry out the evaluation of the antimycobacterial activity of selected eleven medicinal plants. Three different extracts were prepared and evaluated for its antimycobacterial activity against Mycobacterium smegmatis using Mycobacterial Growth Indicator Tube (MGIT) assay. The MGIT assay consists of liquid broth medium that is known to yield better recovery and faster growth of mycobacteria. Isoniazid was used as standard antituberculosis drug. The percentage for anti-mycobacterial smegmatis activity among tested eleven medicinal plants, aqueous extract of Oscimum sanctum, Adhatoda vasica, Leptadenia reticulata and Cocculus hirsutus shows good antimycobacterial activity. Among these four Leptadenia reticulata and Cocculus hirsutus shows potent inhibition as compared to isoniazid. Thus, its result supports the uses of these plants in traditional medicine and also helps to cure and prevent tuberculosis. It can further have studied using more specific methods for antimycobacterial activity.

In vitro Anti-mycobacterial activity of selected medicinal plants against Mycobacterium tuberculosis and Mycobacterium bovis Strains

BMC Complementary and Alternative Medicine, 2013

Background: Tuberculosis (TB) is a global burden with one-third of the world's population infected with the pathogen Mycobacterium tuberculosis complex and annually 1.4 million deaths occur due to the disease. This high incidence of infection and the increased rate of multi-drug resistant and extensively-drug resistant strains of the organism further complicated the problem of TB control and have called for an urgent need to develop new anti-TB drugs from plants. In this study, the in vitro activity of root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis were evaluated against M. tuberculosis and M. bovis strains. Methods: Five Ethiopian medicinal plants, root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis used locally for the management of TB. They were investigated for in vitro antimycobacterial activity against M. tuberculosis and M. bovis strains. 80% methanolic extracts of the plant materials were obtained by maceration. The antimycobacterial activity was determined using 96 wells of microplate with the help of visual Resazurin Microtiter Assay. Results: The crude 80% methanolic extracts of the root of C. aurea, seeds of O. basilicum, and leaves of A. abyssinica, C. macrostachyus, and E. camaldulensis had anti-mycobacterial activity with minimum inhibitory concentration (MIC) ranging from 6.25-100 μg/mL. The MIC of 80% methanol extracts in the order mentioned above ranged 25-100 μg/ml and 12.5-75 μg/mL, 25-100 μg/mL and 25-50 μg/mL, 6.25-50 μg/mL and 12.5-50 μg/mL, 12.5-100 μg/mL and 18.25-50 μg/mL and 6.25-50 μg/mL and 12.5-50 μg/mL, respectively for M. tuberculosis and M. bovis strains. Conclusions: The results support the local use of these plants in the treatment of TB and it is suggested that these plants may have therapeutic value in the treatment of TB. However, further investigations are needed on isolating chemical constituents responsible for eliciting the observed activity in these plants.

Antibacterial effects of Anogeissus leiocarpus (DC.) Guill. Perr. and Terminalia glaucescens Planch. Ex Benth. on rapidly growing mycobacteria species

African Journal of Microbiology Research, 2017

The development of effective and less toxic antimicrobial agents is required for the treatment of respiratory tract infections. This study was carried out to evaluate the phytochemical and antibacterial activities of Anogeissus leiocarpus (DC.) (Guill. & Perr.) and Terminalia glaucescens (Planch. ex Benth.) against non-tuberculous mycobacteria species. The methanol, dichloromethane and aqueous extracts were screened against five (5) non-tuberculosis mycobacteria (NTM) species by agar diffusion method. Minimum inhibitory concentration (MIC) was determined by agar dilution method while bactericidal studies were done by viable count technique. The methanol and aqueous extracts were active against all the test organisms with zones of inhibition ranging from 10±0.0 to 25±0.5 mm. The MIC and MBC range from 0.3125 to 2.5 and 1.25 to 10 mg/mL, respectively. Bactericidal activities of aqueous extracts against Mycobacterium smegmatis ATCC 19420 revealed a drastic dose-dependent decline in the surviving population after 6 h of exposure accompanied by a total (100%) kill after 24 h of exposure. The antimicrobial activities demonstrated by these plants suggest the presence of therapeutically important antimycobacterial compounds and thus justify as well as support the use of these medicinal plants for the treatment of respiratory tract infections.

Potential of plant extracts against Mycobacterium tuberculosis: an integrative review

Peer Review, 2023

Mycobacterium tuberculosis (MTB) is the main cause of tuberculosis. For decades, new forms of treatment and new ways of dealing with the growing resistance acquired by this bacterium to first-line drugs have been studied. Medicinal plants are a source of new bioactive compounds with antimicrobial potential. Objective: to investigate which plant species have already been tested and which main secondary metabolites are active against MTB. Methodology: an integrative review that included in vitro experimental studies, carried out around the world, that used medicinal plant extracts to evaluate the antimycobacterial activity by microdilution, with identification of major compounds, against MTB, between 2011 and 2021, the Web of Science and PubMed were used and the descriptors "medicinal plants AND against AND MTB". Results: 20 species of plants with antimycobacterial activities were found. Four stood out with MIC<10 µg/mL. The variety of secondary metabolites was determinant for antimycobacterial activity, highlighting alkaloids, terpenes and phenolic compounds. Conclusion: the number of secondary metabolites obtained in the extraction is decisive in the antimycobacterial activity.

Screening of Central and South American plant extracts for antimycobacterial activity by the Alamar Blue test

Revista Brasileira de Farmacognosia, 2006

Antimycobacterial activity of medicinal plants from Mozambique

Planta Medica, 2009

Ethnopharmacological relevance: Tuberculosis (TB) is an infectious disease mainly caused by Mycobacterium tuberculosis (Mtb), which generates 9 million new cases worldwide each year. The Mayo ethnicity of southern Sonora, Mexico is more than 2,000 years old, and the Mayos possess extensive knowledge of traditional medicine. Aims of the study: To evaluate the antimycobacterial activity levels of extracts of medicinal plants used by the Mayos against Mtb and Mycobacterium smegmatis (Msm) in the treatment of TB, respiratory diseases and related symptoms. Materials and methods: A total of 34 plant species were collected, and 191 extracts were created with n-hexane, dichloromethane, ethyl acetate (EtOAc), methanol and water. Their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined against Mtb H37Rv using the microplate alamar blue assay (MABA) and against Msm using the resazurin microplate assay (REMA) at 6 and 2 days of exposure, respectively, and at concentrations of 250-1.9 µg/mL (n-hexane extracts) and 1000-7.81 µg/mL (extracts obtained with dichloromethane, EtOAc, methanol and water). Results: Rhynchosia precatoria (Willd.) DC. (n-hexane root extract), Euphorbia albomarginata Torr. & A. Gray.

In vitro anti-mycobacterial activity of selected medicinal plants

2014

Background: Tuberculosis (TB) is a global burden with one-third of the world's population infected with the pathogen Mycobacterium tuberculosis complex and annually 1.4 million deaths occur due to the disease. This high incidence of infection and the increased rate of multi-drug resistant and extensively-drug resistant strains of the organism further complicated the problem of TB control and have called for an urgent need to develop new anti-TB drugs from plants. In this study, the in vitro activity of root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis were evaluated against M. tuberculosis and M. bovis strains. Methods: Five Ethiopian medicinal plants, root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis used locally for the management of TB. They were investigated for in vitro antimycobacterial activity against M. tuberculosis and M. bovis strains. 80% methanolic extracts of the plant materials were obtained by maceration. The antimycobacterial activity was determined using 96 wells of microplate with the help of visual Resazurin Microtiter Assay. Results: The crude 80% methanolic extracts of the root of C. aurea, seeds of O. basilicum, and leaves of A. abyssinica, C. macrostachyus, and E. camaldulensis had anti-mycobacterial activity with minimum inhibitory concentration (MIC) ranging from 6.25-100 μg/mL. The MIC of 80% methanol extracts in the order mentioned above ranged 25-100 μg/ml and 12.5-75 μg/mL, 25-100 μg/mL and 25-50 μg/mL, 6.25-50 μg/mL and 12.5-50 μg/mL, 12.5-100 μg/mL and 18.25-50 μg/mL and 6.25-50 μg/mL and 12.5-50 μg/mL, respectively for M. tuberculosis and M. bovis strains. Conclusions: The results support the local use of these plants in the treatment of TB and it is suggested that these plants may have therapeutic value in the treatment of TB. However, further investigations are needed on isolating chemical constituents responsible for eliciting the observed activity in these plants.