Raw Plant Extracts Research Articles

Metabolite variations in sulla shoots induced by drought stress

AbstractBACKGROUNDClimate change results in an increased occurrence of drought periods, which affect the performance of Mediterranean legumes such as sulla. To evaluate the effects of drought stress on the antioxidant capacity and the accumulation of phytochemicals in sulla shoots, rainfed and drought‐stressed plants (RAI) and watered plants with timely vegetative regeneration (STI) were cultivated in parallel in a field experiment and sampled at four different times.RESULTSDrought stress strongly and positively affected the antioxidant capacity and the accumulation of polyphenols in sulla shoots, with key differences at wintertime. These findings were supported through untargeted and targeted metabolomics of RAI and STI samples, which detailed the accumulation of about 300 secondary metabolites in RAI, mostly polyphenols and saponins.CONCLUSIONThis study provided new insights into the changes in secondary metabolites in sulla due to drought stress, which can be used to produce forages or raw plant extracts with enhanced biological effects. © 2024 Society of Chemical Industry.

ENVIRONMENTAL ETHICS AND ANIMAL WELFARE

This article traces the professional historical trajectory of promoting health and well-being through investigations and interventions in environmental health, associated with education for university, intersectoral and interdisciplinary sustainability based on the vitalist philosophy of Samuel Hahnemann and determinations established by the Brazilian Ministry of Health . Scientific initiation activities developed rational skills for thinking aimed at solving local problems regarding sustainability with raw plant extracts and ultra-diluted and energized solutions for animal welfare. In parallel, there was the construction of competence in environmental ethics in line with the concept of environmental health, the new Brazilian code of medical ethics and the concept of one health. We declare that there are no conflicts of interest.

Effective plant virus enrichment using carbon nanotubes and microfluidics

Plant virus detection and identification in crops is a pillar for disease management, import of crop material, production of clean stock plants and basic plant virology studies. In this report, we present a platform for the enrichment and isolation of known or unknown viruses. This platform is based on carbon nanotube arrays inside a microfluidic device that can be a solution for the identification of low titer viruses from plants. Using our microfluidic devices, we achieved enrichment of two economically important viruses, the orthotospovirus, tomato spotted wilt orthotospovirus (TSWV) and the potyvirus, zucchini yellow mosaic virus (ZYMV). The carbon nanotube arrays integrated in these microfluidic devices are capable of trapping viruses discriminated by their size; the virus rich arrays can be then analyzed by common downstream techniques including immunoassays, PCR, HTS and electron microscopy. This procedure offers a simple to operate and portable sample preparation device capable of trapping viruses from raw plant extracts while reducing the host contamination.

324 Characterization and in Vitro Methane Inhibition of Aloe Vera and Moringa Oleifera Plant Extracts Encapsulated with Alginate Or Alginate-Chitosan Wall Materials Wall Materials

Abstract Although Aloe vera and Moringa oleifera plant extracts are known for their methane inhibition their potential has not been fully exploited in the feeding system of animals due to inconsistent results and loss of efficacy over time. Encapsulation of the plant extract and its bioactive compounds in particular with an inert material will assist to prolong the shelf life of the plant extract. Alginate is a good encapsulant (or a wall material) for encapsulation of plant extracts due to its biocompatibility and low density, among other factors. This study first evaluated A. vera and M. oleifera plant extracts encapsulated with alginate or aligante and chitosan in terms of microscopic morphological characteristics and particle density, and thereafter quantified the potential of encapsulated A. vera and M. oleifera plant extracts as dietary additive to inhibit in vitro methane production from Eragrostis curvula hay. An electron microscope scanning of the freeze-dried alginate or alginate encapsulated plant extracts of A. vera and M. oleifera revealed morphologically spongy and skin-like appearance while for chitosan a scatter particle distribution of different sizes with irregular shape were revealed (Fig. 1). A change in particle density was recorded for the encapsulated plant extracts products compared with the non-encapsulated product due to the inclusion of alginate or alginate-chitosan wall material. The particle density for the encapsulated products was ranging between 4.94 g/10mL (recorded in alginate) to 8.71 g/10mL (recorded in chitosan). During the in vitro gas production study, inclusion of both the encapsulated and non-encapsulated plant extract of A. vera or M. oleifera additives significantly (P < 0.05) reduced methane emission compared with the control group. No adverse effect was observed due to the inclusion of the wall materials in terms of in vitro organic matter digestibility and total gas production (Fig. 2). However, the encapsulated A. vera plant extracts had resulted in a greater methane inhibition of 30.72% (for alginate-extract encapsulated) and 23.09% (for alginate-extract-chitosan encapsulated) against 17.55% reduction recorded for the raw plant extracts. Similarly, the encapsulated M. oleifera plant extracts inhibited methane by 30.02% (alginate-extract encapsulated) and 30.48% (alginate-extract-chitosan encapsulated) against 19.4% recorded for in M. oleifera raw plant extracts. It is revealed that alginate and/or chitosan also had methane reducing effect when tested as additives without the two plant extracts (Table 1,2). However, the differences in particle densities of the micro-particles from the encapsulated and non-encapsulated plant extract additives did not affect their methane inhibition (Fig. 3). Thus, the wall materials (alginate or alginate-chitosan) generally improved the in vitro methane inhibition of encapsulated plant extracts mainly by promoting the inhibition of methane without adverse effect on organic matter digestibility of substrates.

Screening for Bioactive Compounds in Extracts from Leaves, Leafstalks, and Fruits of Ficus Carica

Aqueous, Ethanol extracts and Fig's milk were preliminary phytochemically screened and tested against some pathogenic bacteria. Qualitatively analysis revealed Tannin, Saponin, Flavonoids and Terpenoids and phlobatannins and Steroids gave positive results. Quantitative analysis showed that the highest percentage of alkaloid was 41% in fig milk and 39% in plant leaves. In addition, the crude extracts of fig's milk as well as of the raw plant extracts were tested (using the Disc Diffusion Method) for their antimicrobial activity against the bacterial pathogens. The influences of aqueous and ethanol extracts on some pathogenic: two strains of gram-positive bacteria include Staphylococcus aureus and Streptococcus pneumonia.., and two strains of gram-negative bacteria including Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, Salmonella SPP and Klebsiella aerogenes. , and the sensitivity of the microorganisms to the extracts of plant’s species were compared with each other and with designated antibiotics by measuring the diameter of the inhibition zones, After incubation, the zone of inhibition was measured in mm, a good inhibition of more than 6 mm were observed indicating the effective antibacterial activity of the bioactive compounds in both of the plants leaves extracts and fig's milk. Moreover, the including results of the antibacterial activities of plants leaves extracts and fig's milk were discussed regarding their phytochemical components which exhibited that have a good inhibition zone against six tested bacterial strains.

Biosynthesis and Antimicrobial activities of Silver Nanoparticles (AgNPs) by using Leaf Extracts of Tagetes erecta (Marigold) and Tridax procumbens (Tridax)

In the last few years, it has been seen that the importance of silver nanoparticles has gained much interest by many chemists and biologists. Therefore, Indian medicinal plants has yet to bring numerous sources of profitable, healthy, mostly reduced and stabilized compounds utilized in the biosynthesis of silver nanoparticle. The main aim of this study is to investigate the viable and sustainable ways for the biosynthesis of AgNPs from 1 mM aqueous AgNO3 using leaf extracts of two widely available plants such as Tagetes erecta (Marigold) and Tridax procumbens (Tridax), which are well known for their wide availability and medicinal property. AgNPs were synthesized by the reaction of 1 mM AgNO3 and 5% leaf extracts of each plant separately. The formations of the AgNPs were confirmed by the colour changes of the mixture solution and duly characterized by UV-Visible spectrophotometric analysis. Further, their antibacterial and antifungal activities were tested against two bacterial strains and one fungal strain. Finally, the AgNPs showing better antimicrobial activity was tested for their water disinfection study against three water samples collected from River, Pond and Cannel. Obtained AgNPs from the two different leaf extracts indicated higher antimicrobial activities against Escherichia coli and Bacillus spp. in comparison to both AgNO3 and the raw plant extracts of Marigold and Tridax. The final results showed that both Marigold and Tridax extract silver nanoparticles are showing significant antimicrobial activities, whereas Marigold has little more antimicrobial activity than Tridax. So the bacterial inhibition zone by the silver nanoparticles prepared from the marigold and Tridax leaves extract show maximum inhibition for Gram +ve S. aureus and K. Pnemoniae and Gram negative E. coli as well as A. niger. These synthesised AgNPs when applied for water disinfection, they became a bled to reduce more than 50% of the bacterial growth present in the water samples. At last the synthesized silver nanoparticles were discharged safely in the environment which can be utilized in the processes of pollution remediation.

Solid phase adsorption of emodin on hydrotalcites and inorganic oxides: A preliminary study

Solid phase extraction applied to plant matrices is nowadays a well validated technique allowing the concentration and purification of selected secondary metabolites for subsequent analysis. In this short communication we screened the efficiency of 16 selected solid supports including layered structures (hydrotalcites and zirconium phosphate), magnesium oxide and hydroxide, and finally the phyllosilicates talc and bentonite for the selective concentration of the anthraquinone emodin from raw solid extracts of Polygonum cuspidatum Siebold & Zucc. (sin. Reynoutria japonica Houtt.) (Polygonaceae), commonly known as “Japanese knotweed”. An ethanolic solution of sample extract from this plant was vigorously mixed with fixed quantities of each solid support. Subsequent HPLC analysis, coupled to photodiode array detection, revealed that, among the solid supports assayed, the hydrotalcite zinc aluminum oleate and magnesium oxide were largely the most effective to this concern. Both were able to extract emodin from the raw extract in percentages of 81.5 % and 92.4 %, respectively. The application of the title supports for the extraction and concentration in the solid phase of anthraquinones from raw plant extracts have been reported herein for the first time.

Chemical Composition and Antimicrobial Activity of Bark and Leaf Extracts of Cupressus sempervirens and Juniperus phoenicea Grown in Al- Jabel Al-Akhdar Region, Libya

Background:Plant extracts are important products in the world and have been widely used for isolation of important biologically active products. Because of their significant environmental impact, extensive research has been explored to determine the antimicrobial activity of plant extracts.Methods:Acetone extracts of the bark and leaf of Cupressus sempervirens and Juniperus phoenicea, collected from three different altitudes (125, 391, and 851 m high of sea level) at Al- Jabel Al-Akhdar area, Libya were obtained and analyzed by GC/MS. The antimicrobial activity of the extracts was further evaluated against plant bacteria Rhizobium radiobacter, Erwinia carotovora, Rhodococcus fascians and Ralstonia solanacearum and fungus Botrytis cinerea.Results:The impact of the altitude from the sea level on the quantity and chemical constituents of the extracts was investigated. The yield was largely dependent on tree species and the highest yield (6.50%) was obtained with C. sempervirens L bark of altitude III (851 m of the sea level), while the lowest (1.17%) was obtained with the leaf extract of C. sempervirens L from altitude I (125 m). The chemical composition analyzed by GC/MS confirmed that the leaf extracts of C. sempervirens and J. phoenicea contained a complex mixture of monoterpene hydrocarbons, sesquiterpenes, diterpenes, diterpenoids, terpenophenolic, steroids and phthalates. However, the bark extracts of both trees contained a mixture of sesquiterpenes, diterpenes, diterpenoids, terpenophenolics, phthalates, retinol and steroids. These constituents revealed some variability among the extracts displaying the highest interesting chemotype of totarol (terpenophenolic) in all extracts (14.63-78.19% of the total extract). The extracts displayed a noteworthy antifungal potency with varying degrees of inhibition of growth with EC50 values ranged from 78.50 to 206.90 mg/L. The extracts obtained from the leaves of C. sempervirens showed that the highest inhibitory activity was obtained with the extract of altitude II (391 m) with MIC 565, 510, 380 and 710 mg/L against E. carotovora, R. fascians, and R. radiobacter and R. solanacearum, respectively.Conclusion:Based on antimicrobial activity, raw plant extracts can be a cost-effective way to protect crops from microbial pathogens. Because plant extracts contain several antimicrobial compounds, the development of resistant pathogens can be delayed.

Hitze stabilisiert Malariaimpfstoffkandidaten in komplexen Proteinextrakten

Plants can be an alternative to mammalian cell cultures in terms of vaccine production due to low upstream production costs and a good safety profile. However, product purification can be challenging because of abundant host cell proteins (HCPs) in raw plant extracts. Here we report the optimization of blanching, a heat-based method, that allowed to remove over 90 percent of HCPs and to stabilize a malaria vaccine candidate by carefully adjusting the temperature in the step and combined it with solid-liquid separation and chromatographic purification.

Expression and purification of human phosphatase and actin regulator 1 (PHACTR1) in plant-based systems

Cardiovascular diseases are a prevalent cause of morbidity and mortality especially in industrialized countries. The human phosphatase and actin regulator 1 (PHACTR1) may be involved in such diseases, but its precise regulatory function remains unclear due to the large number of potential interaction partners. The same phenomenon makes this protein difficult to express in mammalian cells, but it is also an intrinsically disordered protein that likely aggregates when expressed in bacteria due to the absence of chaperones. We therefore used a design of experiments approach to test the suitability of three plant-based systems for the expression of satisfactory quantities of recombinant PHACTR1, namely transient expression in tobacco (Nicotiana tabacum) BY-2 plant cell packs (PCPs), whole N. benthamiana leaves and BY-2 cell lysate (BYL). The highest yield was achieved using the BYL: up to 120 mg product kg−1 biomass equivalent within 48 h of translation. This was 1.3-fold higher than transient expression in N. benthamiana together with the silencing inhibitor p19, and 6-fold higher than the PCP system. The presence of Triton X-100 in the extraction buffer increased the recovery of PHACTR1 by 2–200-fold depending on the conditions. PHACTR1 was incompatible with biomass blanching and was stable for less than 16 h in raw plant extracts. Purification using a DDK-tag proved inefficient whereas 15% purity was achieved by immobilized metal affinity chromatography.

Actividad larvicida de extractos vegetales de la familia Asteraceae y modelación matemática para su uso en el control de poblaciones de Aedes aegypti

Dengue, chikungunya and Zika, all transmitted by Aedes aegypti, are diseases that affect the human world population. The evaluation of raw plant extracts allows the establishment of efficient products for the control of populations of this mosquito. This work evaluated the larvicidal activity of 23 species of the family Asteraceae, and its preliminary phytochemical composition, to control populations of A. aegypti. The vegetable material was collected in the department of Quindío, Colombia. We prepared the plant extracts used in the phytochemical characterization and bioassays with the material collected. For each extract, a dose-response bioassay was performed with larvae from the municipality of Armenia (Quindío, Colombia) following the WHO protocol. These assays indicated that after 48h the extracts of Jaegeria hirta (694.8% ± 149.9), Austroeupatorium inulaefolium (753.3% ± 198.8) and Heliopsis oppsitifolia (764.4% ± 170.0) require less concentration to kill 95% of larvae. Additionally, a mathematical model that describes the behavior of the mosquito populations was constructed, in order to evaluate different control strategies using the extracts; the simulations obtained from the numerical solution of the system allow us to conclude that the application of extracts from these plants constitutes a viable tool for A. aegypti control. On the other hand, the preliminary phytochemical analysis of the 23 plant species shows the presence of tannins, quinones, falvonoides, sterols, coumarins and alkaloids. It is concluded then, that J. hirta, A. inulaefoliu and H. oppsitifolia deserve to be further studied, given their larvicidal potential for the control of A. aegypti.

Blanchieren vereinfacht die Reinigung rekombinanter Proteine aus Pflanzen

Plants can be an alternative to mammalian cell cultures in terms of recombinant protein expression due to low upstream production costs and a good safety profile. However, product purification can be challenging due to the abundance of host cell proteins (HCPs) in raw plant extracts and which have to be removed. Here we report different heat treatment methods, e. g. based on leaf blanching. This can facilitate subsequent downstream processing, and thus has the potential to reduce production costs.

Antibacterial Activity of Silver Nanoparticles Synthesized by Using Extracts of Hedera helix

Background: Silver nanoparticles (AgNPs) are one of the most widely applicable particles whose application is increasing in Nano world daily. Silver nanoparticles have expressed significant advances owing to wide range of applications in the field of bio-medical, sensors, antimicrobials, catalysts, electronics, optical fibers, agricultural, bio-labeling and the other areas. Green synthesis is the safe and easiest method of producing silver nanoparticles. Because of the production of the silver ions, silver nanoparticles are found to have the antibacterial activity. Objectives: The aim of this study was to investigate antibacterial activity of silver nanoparticles synthesized by using extracts of Hedera helix against Bacillus subtilis and Klebsiella pneumoniae. Methods: In this experimental study AgNPs were prepared by the reaction of 1mM silver nitrate and extracts of Hedera helix. Antibacterial activity of AgNPs was assessed by using disc diffusion method against Bacillus subtilis and Klebsiella pneumoniae. The AgNPs were characterized by UV-visible (vis) spectrophotometer, particle size analyzer by dynamic light scattering (DLS) method, transmission electron microscopy (TEM). Results: AgNPs obtained showed significantly higher antimicrobial activities against B. subtilis and K. pneumonia in comparison to both AgNO3 and raw plant extracts. Conclusions: Biological methods are a good competent for the chemical procedures, which are environment friendly and convenient.

Antiplasmodial and Cytotoxic Activity of Raw Plant Extracts as Reported by Knowledgeable Indigenous People of the Amazon Region (Vaupés Medio in Colombia).

The in vitro antiplasmodial activity of 122 raw extracts prepared in ethanol and water from 35 medicinal plants reported by the Cubeo indigenous village of the Amazon region (Vaupés Medio in Colombia) was evaluated for efficacy against 3D7 (sensitive to chloroquine) and FCR-3 (resistant to chloroquine) Plasmodium falciparum strains. Five percent of these extracts presented a significant antiplasmodial activity (< 5 µg/mL) and 83 % of them were not cytotoxic. These findings highlight the importance of investigating traditional medicinal plants implemented by these ancestral communities of the Amazon region as well as their potential to become a source of new drugs against malaria.

Leaf extract mediated green synthesis of silver nanoparticles from widely available Indian plants: synthesis, characterization, antimicrobial property and toxicity analysis

Abstract Background In recent years, green synthesis of silver nanoparticles (AgNPs) has gained much interest from chemists and researchers. In this concern, Indian flora has yet to divulge innumerable sources of cost-effective non-hazardous reducing and stabilizing compounds utilized in preparing AgNPs. This study investigates an efficient and sustainable route of AgNP preparation from 1 mM aqueous AgNO3 using leaf extracts of three plants, Musa balbisiana (banana), Azadirachta indica (neem) and Ocimum tenuiflorum (black tulsi), well adorned for their wide availability and medicinal property. Methods AgNPs were prepared by the reaction of 1 mM silver nitrate and 5% leaf extract of each type of plant separately. the AgNPs were duely characterized and tested for their antibacterial activity and toxicity. Results The AgNPs were characterized by UV-visible (vis) spectrophotometer, particle size analyzer (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy-dispersive spectroscopy (EDS). Fourier transform infrared spectrometer (FTIR) analysis was carried out to determine the nature of the capping agents in each of these leaf extracts. AgNPs obtained showed significantly higher antimicrobial activities against Escherichia coli (E. coli) and Bacillus sp. in comparison to both AgNO3 and raw plant extracts. Additionally, a toxicity evaluation of these AgNP containing solutions was carried out on seeds of Moong Bean (Vigna radiata) and Chickpea (Cicer arietinum). Results showed that seeds treated with AgNP solutions exhibited better rates of germination and oxidative stress enzyme activity nearing control levels, though detailed mechanism of uptake and translocation are yet to be analyzed. Conclusion In totality, the AgNPs prepared are safe to be discharged in the environment and possibly utilized in processes of pollution remediation. AgNPs may also be efficiently utilized in agricultural research to obtain better health of crop plants as shown by our study.

Influence of the extraction mode on the yield of hyperoside, vitexin and vitexin‐2′′‐O‐rhamnoside from Crataegus monogyna Jacq. (hawthorn)

The extract of Crataegus monogyna shows sedative, hypotensive, vasodilator and cardio-tonic actions. Although several papers dealing with the extraction of metabolites from Crataegus have been published, the plant productivity in terms of bioactive compounds is not easily understandable as yet. To investigate the influence of the extraction mode on the yield of bioactive compounds from Crataegus monogyna Jacq. in order to evaluate plant productivity. Samples were prepared by extraction of powdered material obtained from top branches, flowers and leaves. Soxhlet extraction, maceration and ultrasound- and microwave-assisted extraction at different experimental conditions were investigated for the exhaustive extraction of hyperoside, vitexin and vitexin-2''-O-rhamnoside. The phytocomponents were identified and quantified by HPLC-UV/PAD, comparing HPLC retention times and UV spectra of individual peaks with those of the standards analysed under the same conditions. An easy-to-use HPLC isocratic method suitable for the quantification of hyperoside, vitexin and vitexin-2''-O-rhamnoside in raw plant extracts was developed. The optimised HPLC methodology was applied to evaluate different extraction procedures. The ultrasound and microwave-assisted extraction protocols showed higher extraction efficiency than the others. In particular, the optimised microwave protocol gave rise to the highest extraction efficiency with high reproducibility. A microwave protocol combined with isocratic HPLC analysis is proposed for the rapid screening of plant materials collected in different environmental conditions in order to evaluate the productivity of Crataegus monogyna Jacq. and to find out the best ecological conditions to cultivate hawthorn in Northern Italy.

High‐throughput direct‐infusion ion trap mass spectrometry: a new method for metabolomics

A fast method was developed to directly infuse raw plant extracts into a linear ion trap mass spectrometer, using the ion trap to isolate and fragment as many ions as possible from the extract. The full mass spectra can be analysed by multivariate statistics to determine discriminating ions, and the fragmentation data allows rapid classification or identification of these ions. The methodology was used to screen a wide range of strains of endophytic fungi in perennial ryegrass seeds for differences in metabolic profiles. The results show that this newly developed methodology is able to determine discriminating ions that can be present in very low concentrations. It also yielded sufficient fragmentation data to classify or identify the discriminating ions.

Amino acids in loose silky-bent ( Apera spica-venti (L.) Beauv.) responding to prosulfocarb exposure and the correlation with physiological effects

Eleven free amino acids were identified in the grass weed loose silky-bent ( Apera spica-venti) after exposure to four rates of the thiocarbamate herbicide prosulfocarb. A new HPTLC method was developed for quantification of amino acids in raw plant extracts. A pattern of significant changes in the content of glutamine, glycine, alanine, and proline due to exposure were detected. The content of all four amino acids increased at increasing exposure rates. Quantitative analyses 7 and 21 days after herbicide application showed that over time the amount of proline increased, the alanine and glycine content decreased, and that at high exposure rates the amount of glutamine increased. Visual effects were evaluated and the biomass was measured. The visual symptoms increased and the biomass decreased with increasing exposure rates. A significant correlation between the changes in the content of the four amino acids and the visual effects and the biomasses were found.

Confirmative assay and quantitative determination ofHedera Helix L. saponins (α-hederin, hederacosides B and C) in raw plant extracts and in cosmetic formulations by EI and CAD MIKE spectrometry

The current methods for the qualitative and quantitative control of cosmetologically active triterpenic saponins in raw plant extracts and in cosmetic formulations are far from satisfactory in terms of specificity and sensitivity. Here we describe a method for the simultaneous detection and quantification of Hedera helix L. saponins directly using crude extracts from plant and cosmetic matrices. The first is achieved by conventional EI-mass spectral analysis (direct DEI conditions); the second by CAD MIKE spectrometry (collisionally activated decomposition mass-analysed ion kinetic energy spectrometry) of the EI-induced fragmentation product at m/z 248, which is common to the three saponins of Hedera helix L. (α-hederin, hederacosides B and C). The method is sensitive (sub ppb level), specific and since it is rapid it might be conveniently employed for the routine quality control of these active constituents in plant extracts and in cosmetic formulations.

Plant screening by chemical and chromatographic procedures under field conditions

The plant kingdom constitutes an invaluable source of new chemical products which may be important due to their biological properties and in particular because of their potential use in medicine. In many countries, mainly in the Tropics, systematic surveys of plants are currently in progress. It is therefore important to have a simple and universal method available for preliminary chemical screening of plants under field conditions. The procedure suggested here is based on the identification in raw plant extracts of some chemical groups of substances (alkaloids, quinones, saponins, polyphenols, anthranoids) by precipitation and colour reactions. To obtain more accurate results, a second stage, based on thin-layer chromatographic separation of extracts of plant material, can be applied. Solvent systems, specific spray reagents and reference substances are proposed. A number of groups of products, including flavonoids, coumarins, cardenolides and steroids can be rapidly identified in plants by this method.