Artemisiae argyi(A.argyi)is a traditional medical plant,with medicinal and economic value.It has been used to treat diseases for more than 2000 years,and its application has been expanded in recent years with the in-d...Artemisiae argyi(A.argyi)is a traditional medical plant,with medicinal and economic value.It has been used to treat diseases for more than 2000 years,and its application has been expanded in recent years with the in-depth research on the chemical composition and pharmacology.It is mainly used clinically for the treatment of“cold pain in the abdomen,cold clotting in the menstrual channels,cold infertility in the uterus,fetal movement,vomiting blood,epistaxis,pus and blood dysentery,and external treatment of itchy skin”and other symptoms.This paper reviews the research on the biological activity and chemical composition of A.argyi by scholars in recent decades.展开更多
A study was conducted to determine the antimicrobial activities of essential oil from Artemisiae argyi leaves. The sample of the essential oil was analyzed by GC-MS. From 18 compounds representing the oils, Eucalypto...A study was conducted to determine the antimicrobial activities of essential oil from Artemisiae argyi leaves. The sample of the essential oil was analyzed by GC-MS. From 18 compounds representing the oils, Eucalyptole (18.42%), Spathulenol (14.32), 4-Methyl-1-(1-methylethyl)-3-cyclohexen-1-ol (3.10%), 3-Carene (2.64%) appeared as the main components. The screening of antimicrobial activity of the essential oil was evaluated using agar diffusion and broth microdilution methods. Gram-positive bacterial were more sensitive than gram-negative bacterial of the 8 microorganisms, and Staphylococcus aureus ATCC 6538 showed the lowest MIC (0.3125%) and MBC (0.625%). In the disc diffusion assay, Staphylococcus epidermidis ATCC 49134 and Bacillus subtilis ATCC 6633 showed obvious inhibitory activity. Survival curve showed that, 2MIC ofArtemisiae argyi essential oil had a lethal effect on Candida albicans within the first 1 h. Results presented here suggest that the essential oil of Artemisiae argyi leaves possesses antimicrobial properties, and provides scientific foundations for exploition ofArtemisiae argyi.展开更多
[Objectives]To obtain the optimal extraction process parameters of volatile oil of Artemisiae Scopariae Herba.[Methods]The controlled variable method was used to take the extraction amount of volatile oil as the evalu...[Objectives]To obtain the optimal extraction process parameters of volatile oil of Artemisiae Scopariae Herba.[Methods]The controlled variable method was used to take the extraction amount of volatile oil as the evaluation indicator,and the effects of single factor solid-to-liquid ratio,soaking time and extraction time on the yield rate of volatile oil were investigated.[Results]When the solid-to-liquid ratio reached 1∶12,the soaking time reached 3 h,and the extraction time reached 2 h,the yield of volatile oil was the highest.[Conclusions]The conditions of this process are stable and feasible,and it is expected to provide a certain reference for extracting volatile oil from Artemisiae Scopariae Herba.展开更多
Objective:To comprehensively review the literature relating to the use of Artemisiae Argyi Folium (AAF;the dried leaf of Artemisia argyi Lévl.et Vant.from the Asteraceae family),and qi ai (AAF grown in Qichun Cou...Objective:To comprehensively review the literature relating to the use of Artemisiae Argyi Folium (AAF;the dried leaf of Artemisia argyi Lévl.et Vant.from the Asteraceae family),and qi ai (AAF grown in Qichun County and the adjacent areas in Hubei Province,China) in the field of traditional Chinese medicine.Furthermore,this study aimed to give results of the contents determination of three bioactive components in qi ai and AAF.Methods:The literature was reviewed to acquire relevant information regarding the quality and application of AAF and qi ai.In addition,the contents of the bioactive components (essential oil,total flavonoids and tannins) were determined in 29 AAF samples collected from China and Korea using the methods in the Chinese pharmacopoeia (2015) or determined by our experiments.Results:The dried leaf of A.argyi Lévl.et Vant.from the Asteraceae family is a mainstream commodity of AAF.Qi ai is a commercial type of AAF well-known for its good quality,which has been recorded in Chinese ancient literature.Modern research has showed that more intense heat with stronger penetration was generated in moxibustion therapy when qi ai moxa stick was burnt compared with general moxa stick,which led to a better curative effect.Our research also showed that the contents of essential oil,total flavonoids and tannin in qi ai were significantly higher than those of general AAF samples.Conclusion:The superior quality of qi ai compared with general AAF may be due to relatively higher contents of bioactive components.This study provides basic information for further study and utilization of AAF and qi ai.展开更多
Background:Artemisia vulgaris,a medicinal aromatic plant,is widely used as a food item,tonic pharmaceutical,and cosmetic industry additive owing to its antibacterial,antihypertensive,hepatoprotective,antioxidant,and a...Background:Artemisia vulgaris,a medicinal aromatic plant,is widely used as a food item,tonic pharmaceutical,and cosmetic industry additive owing to its antibacterial,antihypertensive,hepatoprotective,antioxidant,and antispasmodic properties.But the effect of different geographic locations on the chemical composition and bioactivities of its extracts is unclear.Methods:Biological activities of essential oils and ethanol extracts of three varieties of Artemisia vulgaris leaves,which are grown in Shanxi province China,were studied.Results:Gas chromatography-mass spectrometry analysis revealed that the main components of essential oils were terpenes and ketones.Essential oils and ethanol extract of Artemisia vulgaris leaves possessed good antioxidant activities,and their half maximal inhibitory concentrations determined using 1,1-diphenyl-2-picrylhydrazyl and 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate)assays were 57.0 and 22.9μg/mL,respectively.The essential oils also exhibited remarkable antibacterial activity against three foodborne pathogenic bacterial strains.The ethanol extract presented a high anticancer activity against the MGC-803 human gastric cancer cell line.Conclusion:These biological activities were well correlated with the composition of the extract and EOs,which in turn is affected by the genetic composition of Artemisia vulgaris and geographic location and diverse climatic condition under which it is grown.These findings demonstrate the remarkable potential of Artemisia vulgaris as a valuable source of antioxidant,antibacterial,and anticancer agents.展开更多
[Objectives]This paper was to figure out whether the dominant bacterial community has the role and effect of bacterial community and its defense mechanism against potential pathogenic fungi in Artemisia annua,and thus...[Objectives]This paper was to figure out whether the dominant bacterial community has the role and effect of bacterial community and its defense mechanism against potential pathogenic fungi in Artemisia annua,and thus establish a systematic model of bacteria-fungus-plant.[Methods]Fifty-eight strains of bacteria and one strain of pathogenic fungi,Globisporangium ultimatum,were used for the experiments.These 58 bacterial strains were assembled into a bacterial community,and the bacteria with abundance in the top 1%were reassembled into a dominant bacterial community as measured by 16S rDNA.[Results]The growth of A.annua seedlings inoculated with bacterial communities and pathogenic fungi or dominant bacterial communities and pathogenic fungi was significantly better than that of A.annua seedlings inoculated with pathogenic fungi during in vitro confrontation,which was evident in both enzymatic and non-enzymatic antioxidant assays.[Conclusions]The results suggest that the dominant bacterial community has a crucial role as a representative core microbial community of synthetic bacterial community,which can protect plants by interfering with the growth of phytopathogenic fungi mediated by chemical signals,and can be used as the main synthetic community of biocides to achieve the effect of biocontrol.展开更多
Individuals with Glucose-6-phosphate dehydrogenase (G6PD) deficiency are susceptible to hemolytic anemia when exposed to pro-oxidant substances. This study investigates the hemolytic impact of Artemisia annua (A. annu...Individuals with Glucose-6-phosphate dehydrogenase (G6PD) deficiency are susceptible to hemolytic anemia when exposed to pro-oxidant substances. This study investigates the hemolytic impact of Artemisia annua (A. annua) extracts in G6PD-deficient subjects through a mixed experimental approach. In the in vitro phase, red blood cells from G6PD-deficient individuals and rats induced with Dehydroepiandrosterone (DHEA) were exposed to various concentrations of A. annua infusion, with distilled water and physiological saline as positive and negative controls respectively. The in vivo study involved G6PD-deficient Wistar rats divided into three groups receiving A. annua infusion, quinine (positive control), and distilled water (negative control) via gavage. Blood samples were collected for biochemical and hematological analyses. Notably, at a 40% concentration of A. annua infusion, there was a significant increase in the hemolysis rate of G6PD-deficient red blood cells compared to controls (p A. annua exhibited elevated aspartate aminotransferase (129.25 ± 4.55 U/L vs. 80.09 ± 4.03 U/L;p A. annua infusion tested positive for saponins. These findings underscore the risk of hemolysis in G6PD-deficient individuals upon ingesting A. annua.展开更多
All nuclei in mesophyll cells of Artemisia marschalliana are located in vacuoles and occupy up to 90% of their volume. The ultrastructural organization of chromatin in nuclei shows different degrees of its decondensat...All nuclei in mesophyll cells of Artemisia marschalliana are located in vacuoles and occupy up to 90% of their volume. The ultrastructural organization of chromatin in nuclei shows different degrees of its decondensation, up to complete separation of DNA from histones. It is possible that the separation of DNA from histones enables Artemisia to grow in soils with high salinity.展开更多
Maintaining the stability of exotic sand-binding shrub has become a large challenge in arid and semi-arid grassland ecosystems in northern China.We investigated two kinds of shrublands with different BSCs(biological s...Maintaining the stability of exotic sand-binding shrub has become a large challenge in arid and semi-arid grassland ecosystems in northern China.We investigated two kinds of shrublands with different BSCs(biological soil crusts)cover in desert steppe in Northwest China to characterize the water sources of shrub(Caragana intermedia Kuang et H.C.Fu)and grass(Artemisia scoparia Waldst.et Kit.)by stable 18O isotopic.Our results showed that both shrublands were subject to persistent soil water deficiency from 2012 to 2017,the minimum soil depth with CV(coefficient of variation)<15% and SWC(soil water content)<6% was 1.4 m in shrubland with open areas lacking obvious BSC cover,and 0.8 m in shrubland covered by mature BSCs.For C.intermedia,a considerable proportion of water sources pointed to the surface soil.Water from BSCs contributed to averages 22.9%and 17.6%of the total for C.intermedia and A.scoparia,respectively.C.intermedia might use more water from BSCs in rainy season than dry season,in contrast to A.scoparia.The relationship between shrub(or grass)and soil water by δ^(18)O shown significant differences in months,which partly verified the potential trends and relations covered by the high variability of the water source at seasonal scale.More fine roots at 0-5 cm soil layer could be found in the surface soil layer covered by BSCs(8000 cm/m^(3))than without BSCs(3200 cm/m^(3)),which ensured the possibility of using the surface soil water by C.intermedia.The result implies that even under serious soil water deficiency,C.intermedia can use the surface soil water,leading to the coexistence between C.intermedia and A.scoparia.Different with the result from BSCs in desert areas,the natural withdrawal of artificial C.intermedia from desert steppe will be a long-term process,and the highly competitive relationship between shrubs and grasses also determines that its habitat will be maintained in serious drought state for a long time.展开更多
Artemisia argyi is a traditional Chinese herb,which is widely distributed in various areas in China.In recent years,due to the development of A.argyi related industries and the continuous improvement of people’s heal...Artemisia argyi is a traditional Chinese herb,which is widely distributed in various areas in China.In recent years,due to the development of A.argyi related industries and the continuous improvement of people’s health awareness,experts and scholars are concerned about the nutrients and effects of A.argyi.A.argyi is rich in carbohydrates,proteins,essential amino acids,fatty acids,minerals,vitamins,dietary fibers,flavonoids and other nutrients.It has the effects of antioxidant,anti-tumor,antibacterial,antiinflammatory,neuroprotection and immune regulations,and has been widely applied in the fields of COVID-19 prevention,food health care,food preservation and feed breeding,and has good development potential.This review summarized the research and application of A.argyi nutrients,in order to provide reference and application for further study.展开更多
The aim of this study was to investigate the chemical composition of the essential oils of “Covid-Organics” of Madagascar (62% Artemisia annua and two other undisclosed medicinal plants) used as curative and prevent...The aim of this study was to investigate the chemical composition of the essential oils of “Covid-Organics” of Madagascar (62% Artemisia annua and two other undisclosed medicinal plants) used as curative and preventive treatments of Covid-19, to identify its constituent species. The essential oils isolated by hydro-distillation from two samples (curative and preventive) were analyzed by GC-FID and GC-MS. These essential oils (curative and preventive) were mainly dominated by camphor (17.9% and 11.9%, respectively), spathulenol (4.8% and 11.8%, respectively), α-acorenol (4.3% and 3.7%, respectively), (E)-β-caryophyllene (3.4% and 4.2%, respectively), 1,8-cineole (3.1% and 3.6%, respectively), hexadecanoïc acid (3.8% and 3.2%, respectively) and caryophyllene oxide (3.4% and 2.4%, respectively). From the chemical composition, two species were identified, A. annua characterised by camphor and Cinnamomumcamphora (Ravintsara) characterised by 1,8-cineole and sabinene. However, we were unable to identify the third species.展开更多
基金This work was financially supported by National Nature Science Foundation of China(81973284)Scientific Research Foundation of the Education Department of Liaoning Province(LJKZ0944).
文摘Artemisiae argyi(A.argyi)is a traditional medical plant,with medicinal and economic value.It has been used to treat diseases for more than 2000 years,and its application has been expanded in recent years with the in-depth research on the chemical composition and pharmacology.It is mainly used clinically for the treatment of“cold pain in the abdomen,cold clotting in the menstrual channels,cold infertility in the uterus,fetal movement,vomiting blood,epistaxis,pus and blood dysentery,and external treatment of itchy skin”and other symptoms.This paper reviews the research on the biological activity and chemical composition of A.argyi by scholars in recent decades.
文摘A study was conducted to determine the antimicrobial activities of essential oil from Artemisiae argyi leaves. The sample of the essential oil was analyzed by GC-MS. From 18 compounds representing the oils, Eucalyptole (18.42%), Spathulenol (14.32), 4-Methyl-1-(1-methylethyl)-3-cyclohexen-1-ol (3.10%), 3-Carene (2.64%) appeared as the main components. The screening of antimicrobial activity of the essential oil was evaluated using agar diffusion and broth microdilution methods. Gram-positive bacterial were more sensitive than gram-negative bacterial of the 8 microorganisms, and Staphylococcus aureus ATCC 6538 showed the lowest MIC (0.3125%) and MBC (0.625%). In the disc diffusion assay, Staphylococcus epidermidis ATCC 49134 and Bacillus subtilis ATCC 6633 showed obvious inhibitory activity. Survival curve showed that, 2MIC ofArtemisiae argyi essential oil had a lethal effect on Candida albicans within the first 1 h. Results presented here suggest that the essential oil of Artemisiae argyi leaves possesses antimicrobial properties, and provides scientific foundations for exploition ofArtemisiae argyi.
文摘[Objectives]To obtain the optimal extraction process parameters of volatile oil of Artemisiae Scopariae Herba.[Methods]The controlled variable method was used to take the extraction amount of volatile oil as the evaluation indicator,and the effects of single factor solid-to-liquid ratio,soaking time and extraction time on the yield rate of volatile oil were investigated.[Results]When the solid-to-liquid ratio reached 1∶12,the soaking time reached 3 h,and the extraction time reached 2 h,the yield of volatile oil was the highest.[Conclusions]The conditions of this process are stable and feasible,and it is expected to provide a certain reference for extracting volatile oil from Artemisiae Scopariae Herba.
文摘Objective:To comprehensively review the literature relating to the use of Artemisiae Argyi Folium (AAF;the dried leaf of Artemisia argyi Lévl.et Vant.from the Asteraceae family),and qi ai (AAF grown in Qichun County and the adjacent areas in Hubei Province,China) in the field of traditional Chinese medicine.Furthermore,this study aimed to give results of the contents determination of three bioactive components in qi ai and AAF.Methods:The literature was reviewed to acquire relevant information regarding the quality and application of AAF and qi ai.In addition,the contents of the bioactive components (essential oil,total flavonoids and tannins) were determined in 29 AAF samples collected from China and Korea using the methods in the Chinese pharmacopoeia (2015) or determined by our experiments.Results:The dried leaf of A.argyi Lévl.et Vant.from the Asteraceae family is a mainstream commodity of AAF.Qi ai is a commercial type of AAF well-known for its good quality,which has been recorded in Chinese ancient literature.Modern research has showed that more intense heat with stronger penetration was generated in moxibustion therapy when qi ai moxa stick was burnt compared with general moxa stick,which led to a better curative effect.Our research also showed that the contents of essential oil,total flavonoids and tannin in qi ai were significantly higher than those of general AAF samples.Conclusion:The superior quality of qi ai compared with general AAF may be due to relatively higher contents of bioactive components.This study provides basic information for further study and utilization of AAF and qi ai.
基金This work was financially supported by the National Natural Science Foundation of China(Grant No.32001817)the Science and Technology Innovation Plan of Colleges and Universities of Shanxi Province(2020L0298)the College student innovation project of North University of China and the start-up funds for scientific research at North University of China(No.304-1101285714).
文摘Background:Artemisia vulgaris,a medicinal aromatic plant,is widely used as a food item,tonic pharmaceutical,and cosmetic industry additive owing to its antibacterial,antihypertensive,hepatoprotective,antioxidant,and antispasmodic properties.But the effect of different geographic locations on the chemical composition and bioactivities of its extracts is unclear.Methods:Biological activities of essential oils and ethanol extracts of three varieties of Artemisia vulgaris leaves,which are grown in Shanxi province China,were studied.Results:Gas chromatography-mass spectrometry analysis revealed that the main components of essential oils were terpenes and ketones.Essential oils and ethanol extract of Artemisia vulgaris leaves possessed good antioxidant activities,and their half maximal inhibitory concentrations determined using 1,1-diphenyl-2-picrylhydrazyl and 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate)assays were 57.0 and 22.9μg/mL,respectively.The essential oils also exhibited remarkable antibacterial activity against three foodborne pathogenic bacterial strains.The ethanol extract presented a high anticancer activity against the MGC-803 human gastric cancer cell line.Conclusion:These biological activities were well correlated with the composition of the extract and EOs,which in turn is affected by the genetic composition of Artemisia vulgaris and geographic location and diverse climatic condition under which it is grown.These findings demonstrate the remarkable potential of Artemisia vulgaris as a valuable source of antioxidant,antibacterial,and anticancer agents.
基金Supported by Science and Technology Plan Project of Guizhou Province,China(QKH JC[2020]1Y179)Key Field Project of Education Department of Guizhou Province(QJHKYZ[2021]044)+1 种基金Forestry Research Project of Guizhou Province(QLKH[2021]11)Project of Guizhou Provincial Characteristic Key Laboratory(QJHKY[2021]002).
文摘[Objectives]This paper was to figure out whether the dominant bacterial community has the role and effect of bacterial community and its defense mechanism against potential pathogenic fungi in Artemisia annua,and thus establish a systematic model of bacteria-fungus-plant.[Methods]Fifty-eight strains of bacteria and one strain of pathogenic fungi,Globisporangium ultimatum,were used for the experiments.These 58 bacterial strains were assembled into a bacterial community,and the bacteria with abundance in the top 1%were reassembled into a dominant bacterial community as measured by 16S rDNA.[Results]The growth of A.annua seedlings inoculated with bacterial communities and pathogenic fungi or dominant bacterial communities and pathogenic fungi was significantly better than that of A.annua seedlings inoculated with pathogenic fungi during in vitro confrontation,which was evident in both enzymatic and non-enzymatic antioxidant assays.[Conclusions]The results suggest that the dominant bacterial community has a crucial role as a representative core microbial community of synthetic bacterial community,which can protect plants by interfering with the growth of phytopathogenic fungi mediated by chemical signals,and can be used as the main synthetic community of biocides to achieve the effect of biocontrol.
文摘Individuals with Glucose-6-phosphate dehydrogenase (G6PD) deficiency are susceptible to hemolytic anemia when exposed to pro-oxidant substances. This study investigates the hemolytic impact of Artemisia annua (A. annua) extracts in G6PD-deficient subjects through a mixed experimental approach. In the in vitro phase, red blood cells from G6PD-deficient individuals and rats induced with Dehydroepiandrosterone (DHEA) were exposed to various concentrations of A. annua infusion, with distilled water and physiological saline as positive and negative controls respectively. The in vivo study involved G6PD-deficient Wistar rats divided into three groups receiving A. annua infusion, quinine (positive control), and distilled water (negative control) via gavage. Blood samples were collected for biochemical and hematological analyses. Notably, at a 40% concentration of A. annua infusion, there was a significant increase in the hemolysis rate of G6PD-deficient red blood cells compared to controls (p A. annua exhibited elevated aspartate aminotransferase (129.25 ± 4.55 U/L vs. 80.09 ± 4.03 U/L;p A. annua infusion tested positive for saponins. These findings underscore the risk of hemolysis in G6PD-deficient individuals upon ingesting A. annua.
文摘All nuclei in mesophyll cells of Artemisia marschalliana are located in vacuoles and occupy up to 90% of their volume. The ultrastructural organization of chromatin in nuclei shows different degrees of its decondensation, up to complete separation of DNA from histones. It is possible that the separation of DNA from histones enables Artemisia to grow in soils with high salinity.
基金This research was funded by the National Natural Science Foundation of China(32060313,31760707).
文摘Maintaining the stability of exotic sand-binding shrub has become a large challenge in arid and semi-arid grassland ecosystems in northern China.We investigated two kinds of shrublands with different BSCs(biological soil crusts)cover in desert steppe in Northwest China to characterize the water sources of shrub(Caragana intermedia Kuang et H.C.Fu)and grass(Artemisia scoparia Waldst.et Kit.)by stable 18O isotopic.Our results showed that both shrublands were subject to persistent soil water deficiency from 2012 to 2017,the minimum soil depth with CV(coefficient of variation)<15% and SWC(soil water content)<6% was 1.4 m in shrubland with open areas lacking obvious BSC cover,and 0.8 m in shrubland covered by mature BSCs.For C.intermedia,a considerable proportion of water sources pointed to the surface soil.Water from BSCs contributed to averages 22.9%and 17.6%of the total for C.intermedia and A.scoparia,respectively.C.intermedia might use more water from BSCs in rainy season than dry season,in contrast to A.scoparia.The relationship between shrub(or grass)and soil water by δ^(18)O shown significant differences in months,which partly verified the potential trends and relations covered by the high variability of the water source at seasonal scale.More fine roots at 0-5 cm soil layer could be found in the surface soil layer covered by BSCs(8000 cm/m^(3))than without BSCs(3200 cm/m^(3)),which ensured the possibility of using the surface soil water by C.intermedia.The result implies that even under serious soil water deficiency,C.intermedia can use the surface soil water,leading to the coexistence between C.intermedia and A.scoparia.Different with the result from BSCs in desert areas,the natural withdrawal of artificial C.intermedia from desert steppe will be a long-term process,and the highly competitive relationship between shrubs and grasses also determines that its habitat will be maintained in serious drought state for a long time.
基金Supported by the Research on Scientific and Technological Innovation of Beef Cattle in Northeast Cold Region by Ministry of Education(002020010)。
文摘Artemisia argyi is a traditional Chinese herb,which is widely distributed in various areas in China.In recent years,due to the development of A.argyi related industries and the continuous improvement of people’s health awareness,experts and scholars are concerned about the nutrients and effects of A.argyi.A.argyi is rich in carbohydrates,proteins,essential amino acids,fatty acids,minerals,vitamins,dietary fibers,flavonoids and other nutrients.It has the effects of antioxidant,anti-tumor,antibacterial,antiinflammatory,neuroprotection and immune regulations,and has been widely applied in the fields of COVID-19 prevention,food health care,food preservation and feed breeding,and has good development potential.This review summarized the research and application of A.argyi nutrients,in order to provide reference and application for further study.
文摘The aim of this study was to investigate the chemical composition of the essential oils of “Covid-Organics” of Madagascar (62% Artemisia annua and two other undisclosed medicinal plants) used as curative and preventive treatments of Covid-19, to identify its constituent species. The essential oils isolated by hydro-distillation from two samples (curative and preventive) were analyzed by GC-FID and GC-MS. These essential oils (curative and preventive) were mainly dominated by camphor (17.9% and 11.9%, respectively), spathulenol (4.8% and 11.8%, respectively), α-acorenol (4.3% and 3.7%, respectively), (E)-β-caryophyllene (3.4% and 4.2%, respectively), 1,8-cineole (3.1% and 3.6%, respectively), hexadecanoïc acid (3.8% and 3.2%, respectively) and caryophyllene oxide (3.4% and 2.4%, respectively). From the chemical composition, two species were identified, A. annua characterised by camphor and Cinnamomumcamphora (Ravintsara) characterised by 1,8-cineole and sabinene. However, we were unable to identify the third species.