Analgesic activity of Heliopsis longipes and its effect on the nervous system
V.G. Cilia-López, B.I. Juárez-Flores, J.R. Aguirre-Rivera & J.A. Reyes-Agüero
Abstract
Heliopsis longipes S.F. Blake (Asteraceae: Heliantheae) (chilcuague) is used in Mexican traditional medicine against parasites and to alleviate tooth and muscle pains. Its biocide effect has already been experimentally demonstrated; however, its analgesic action and its action on the nervous system (NS) have not been investigated yet. The objectives of this study were to evaluate the analgesic action of affinin and the H. longipes root ethanol extract, as well as their effects on the NS using an animal model. The ethanol extract was obtained by maceration, and affinin was purified from it through chromatographic techniques. Chemical and thermal analgesia were used to assess their analgesic proprieties. Irwin’s test was used to evaluate their stimulating or depressing effects. The ethanol extract and affinin displayed analgesic action imilar to ketorolac and stimulating effect comparable to caffeine on the nervous system of adult mice.
Changes in affinin contents in Heliopsis longipes (chilcuague) after a controlled elicitation strategy under greenhouse conditions
IxchelParola-Contrerasa, Erik G.Tovar-Pereza,c, AlejandraRojas-Molinab, Francisco J.Luna-Vazquezb, IrineoTorres-Pachecoa, Rosalia V.Ocampo-Velazqueza, Ramon G.Guevara-Gonz.leza
Abstract
Heliopsis longipes (H. longipes) is an endemic plant of the Sierra Gorda, Mexico, whose root crude extract is used as an analgesic, anti-inflammatory and anti-ulcerative. Affinin is the main substance that provides these properties. Affinin concentrations have been seen to vary depending on the time of the year in which it is collected. Controlled cultivation of H. longipes may reduce the synthesis of affinin but using elicitation technique it might be possible to increase and standardize the biosynthesis of this specialized metabolite. The aim of this research was to determine the changes in the affinin contents in H. longipes roots after controlled elicitation using salicylic acid and hydrogen peroxide as elicitors under greenhouse conditions. After a period of acclimation of 150 days for chilcuague plants, both elicitors were foliarly sprayed for two weeks. Besides affinin contents, changes on the biomass production, catalase, superoxide dismutase, phenylalanine ammonia-lyase and valine decarboxylase enzymatic activities in H. longipes were evaluated. The results showed that low doses of both elicitors significantly increased affinin contents in roots in comparison with controls without affecting morphological variables. This increase in affinin was correlated with concomitant increases in enzymatic activities related to stress responses as catalase (CAT), superoxide dismutase (SOD), phenylalanine ammonia-lyase (PAL), as well as the increase in valine decarboxylase activity (VDC), an enzyme related with biosynthesis of aliphatic alkamides as affinin. Thus, controlled elicitation might be an interesting strategy to increase affinin contents in chilcuague cultivated under greenhouse conditions.
EFFECT OF THE ROOTS EXTRACT FROM Heliopsis longipes on Aspergillus parasiticus GROWTH
John M. Velez-Haro, Nydia E. Buitimea-Cantúa, Ema C. Rosas-Burgos, Jorge Molina-Torres and Génesis V. Buitimea-Cantúa
Abstract
The organic extracts from Heliopsis longipes roots possess interesting biological and pharmacological activities. However, the effect on Aspergillus parasiticus has not been
investigated so far. This study was aimed to evaluate the antifungal effect of the ethanolic extract from H. longipes roots against A. parasiticus growth. Four extract concentrations, 50, 75, 150 and 200 μg/mL were evaluated for antifungal effect, and the anti-aflatoxigenic assay was tested at 150 and 200 μg/mL. The minimal inhibitory concentration that inhibited
50 and 99% of growth (MIC50 and MIC99) were determined. Ethanolic extract was characterized by GC-EIMS analysis and its main bioactive compounds were identified. All tested concentrations inhibit the radial growth of A. parasiticus and the MIC50 and MIC99 were 116.94 and 1593.98 μg/mL, respectively. These results showed the first evidence of the antifungal effect of H. longipes on the radial growth and spore germination rate of this pathogenic fungus. The antifungal activity of H. longipes extract was attributed to affinin (7.24 ± 0.13 mg/g of fresh tissue), the most abundant alkamide detected by GC-EIMS analysis. Although, the extract did not inhibit the aflatoxins´ production, it can be used to prevent A. parasiticus growth before the mycotoxins production occur. Therefore, the extract has potential as natural antifungal agent against A. parasiticus contamination.
Anti-inflammatory effect of an ethanolic root extract of Heliopsis longipes in vitro
Ivones Hernández, Yeny Lemus, Sylvia Priet, Jorge Molina-Torres, Gabino Garrido
Abstract
Heliopsis longipes (A. Gray) Blake (Asteraceae) is a species broadly used in Mexican Traditional Medicine. The present study illustrates the effects of the root ethanolic extract from this species on the production of tumor necrosis factor alpha (TNFα) and nitric oxide (NO) by activated RAW264.7 macrophage. The extract showed an inhibitory activity on TNFα (IC50= 223.0 μg/mL) and NO (IC50 = 136.9 μg/mL). These results represent a contribution to the elucidation of the mechanism involved in the analgesic and anti-inflammatory effects reported for the H. longipes extract.
Antihyperalgesia Induced by Heliopsis longipes Extract
Mario I. Ortiz, Raquel Cariño-Cortés, Nury Pérez-Hernández, Héctor Ponce-Monter, Eduardo Fernández-Martínez, Gilberto Castañeda-Hernández, Iliana I. Acosta-Madrid and Virginia G. Cilia-López
Abstract
Heliopsis longipes is an herbaceous plant found in Mexico. Heliopsis longipes is traditionally used for its analgesic and anesthetic properties. Plant extracts may represent a therapeutic advantage for the clinical treatment of pain. Therefore, the main objective of this study was to determine the possible antihyperalgesic effect produced by the Heliopsis longipes ethanolic extract (HLEE) in the Hargreaves model of thermal hyperalgesia in the mouse. HLEE was administrated systemically to mice and the antihyperalgesic effect was evaluated using the thermal hyperalgesia test. Oral Administration of HLEE produced a dose-dependent antihyperalgesic effect. Previously, it was reported that Heliopsis longipes extract was able to release GABA in mice temporal cortex slices. Therefore, it is likely that the antihyperalgesic effect observed in our study could result from GABA liberation and its inhibition of excessive excitation of nociceptive circuits in the thalamus and cortex evoked by tissue injury. Our results suggest that HLEE may represent a therapeutic advantage for the clinical treatment of inflammatory pain.
Interaction between Heliopsis longipes extract and diclofenaconthe thermal hyperalgesiatest
I.I. Acosta-Madrida, G. Castañeda-Hernández, V.G. Cilia-López, R.Cariño-Cortés, N. Pérez-Hernández, E. Ferná ndez-Martínez, M.I. Ortiz,
Abstract
Heliopsis longipes is an herbaceous plant found in Mexico, used traditionally for its analgesic and anesthetic activities. Plant extracts in combined use with synthetic drugs may represent a therapeutic advantage for the clinical treatment of pain, allowing the use of lower doses, and limiting side-effects. Therefore, the main objective of this study was to determine the possible pharmacological interaction between Heliopsis longipes ethanolic extract (HLEE) and diclofenac in the Hargreaves model of thermal hyperalgesia in the mouse. HLEE, diclofenac or fixed-dose ratio HLEE–diclofenac combinations were administered systemically to mice and the anti hyperalgesic effect was evaluated using the thermal hyperalgesia test. All treatments produced a dose-dependent antihyperalgesic effect. ED30 values were estimated for all the treatments and an isobologram was constructed. The derived theoretical ED30 value for the HLEE–diclofenaccombination was 54.479.4 mg/kg bodywt, significantly higher than the actually observed experimental ED30 value, 8.674.0 mg/kg bodywt. This result corresponds to synergistic interaction between HLEE and diclofenac in the Hargreaves model of thermalhyperalgesia. Data suggest that low doses of the HLEE–diclofenac combination can interact synergistically at the systemic level and that this association may therefore represent a therapeutic advantage for the clinical treatment of inflammatory pain.
Alkamide-rich Heliopsis longipes extract promotes resistance to grey mould disease in ornamental lisianthus
A. Cárdenas-Flores, D.A. Rodríguez-Chávez, A. Flores-Olivas, L. Ibarra-Jiménez and J.H. Valenzuela-Soto
Abstract
Lisianthus is an important flower crop susceptible to Botrytis cinerea grey mould. Therefore we studied whether lisianthus plants could be protected with the use of alkamides from the herb Heliopsis longipes against artificially inoculated B. cinerea during pre- and postharvest. Grey mould symptoms on inoculated leaves of lisianthus were decreased three times by pre-treatment with alkamides, compared to untreated, greenhouse-cultured lisianthus. In postharvest stored shoots no protection was observed. Our results show that the exogenous application of alkamides promoted resistance in lisianthus plants against B. cinerea during preharvest but not postharvest storage.
Phenolic compounds and antioxidant activity of methanolic extracts from leaves and flowers of chilcuague
ID Ixchel Parola-Contreras, ID Ramón G. Guevara-González, ID Ana A. Feregrino-Pérez, ID Rosalía Reynoso-Camacho, ID Iza F. Pérez-Ramírez, ID Rosalía V. Ocampo-Velázquez, ID Alejandra Rojas-Molina, ID Francisco Luna-Vázquez, ID Erik G. Tovar-Pérez
Abstract
Heliopsis longipes root is widely used for its culinary and medicinal importance, yet the foliage is often discarded during collection. To better exploit the plant, it is essential to study the phytochemical content and biological activity present in its various organs, including the foliage. The concentration of phenolic compounds with antioxidant activity varies based on the plant organ and the geographic location of collection. In this study, H. longipes plants were collected from three locations in the Sierra Gorda of Mexico (Conca, San Juan Buenaventura, and Beltran) between 2017 and 2018. Methanolic extracts from the leaves and flowers of H. longipes were analyzed to determine their phenolic content and radical scavenging activity (DPPH• and ABTS•+). Chromatographic characterization (UPLC) further detailed the extracts. The total content of polyphenols, flavonoids, and condensed tannins varied depending on the plant organ and collection site, with Beltran leaves showing the highest levels of phenolic compounds with scavenging activity against DPPH• and ABTS•+. Identified compounds in the extracts included phenolic acids and flavonoids. The study concludes that geographical and climatic conditions significantly influence the phenolic content and antioxidant activity of methanolic extracts from H. longipes leaves and flowers.
Anti-arthritic activity evaluated in a Freund’s adjuvant-induced model in rodents
Carolina Escobedo-Martíneza, Silvia Laura Guzmán-Gutiérrez, María de los Milagros Hernández-Méndeza, Julia Cassanic, Alfonso Trujillo-Valdiviaa, Luis Manuel Orozco-Castellanosa, Raúl G. Enríquez
Abstract
This study assesses the anti-arthritic effect of the affinin-enriched (spilanthol, main alkamide) hexaneextract from the roots of Heliopsis longipes (A. Gray) S.F. Blake, Asteraceae, on a Freund adjuvant-inducedarthritis model in rodents. The extract was orally administered at a dose of 2, 6.6, or 20 mg/kg; a significantedema-inhibitory activity in the acute and chronic phases was observed with a dose of 2 and 20 mg/kg,respectively. The extract showed higher anti-inflammatory and anti-arthritic effects than the referencedrug phenylbutazone (80 mg/kg). Moreover, the extract prevented the occurrence of secondary lesionsassociated to this pharmacological model.
HPLC Determination of the Alkamide Affinin in Fresh and Dry Roots of Heliopsis longipes (Asteraceae) and HS-SPME-GC-MS-TOF Analysis of Volatile Components
María Isabel Aguilar, Nancy E. Castillo, Cristian Alvarado-López, Georgina Duarte-Lisci, Ramiro Ríos-Gómez, María Yolanda Rios
Abstract
Heliopsis longipes (Asteraceae) is a plant whose roots are commonly used in Mexican cuisine as a substitute for hot pepper. This plant is also used in traditional medicine for its therapeutical properties. Previous reports detected the presence of affinin – an alkamide with analgesic properties – in extracts of H. longipes. Here, we describe the development of a high-performance liquid chromatography-UV detector method for the determination of the alkamide affinin (1) as the major component in fresh and dry roots of H. longipes. Compound 1 was quantitatively determined employing a Kromasil 100 C18 3 μm particle size column, using the isocratic mobile phase acetonitrile-water (55+45). The flowrate was 0.8 mL/min and the UV detection was at 213 nm. The limits of detection and quantitation were 0.0009 and 7.6 μg/mL, respectively. Compound 1 showed good linearity in the 75 to 150 μg/mL range; recovery was within 99.3 and 101.9 %. Quantities of affinin measured by this method ranged between 6.9 and 8.5 mg per gram of the dry and fresh roots; thus 1 could be used as marker for H. longipes. The LC method described here proved to be reliable, reproducible, accurate, and could be used for quality control of H. longipes medicinal materials. In addition, its volatile chemical composition is described and was assessed via GC-MS using headspace solid-phase microextraction; compounds were identified by matching against the standard mass spectral database of the National Institute of Standards and Technology (NIST). Twenty-eight recognized compounds represent 99.9 % of the total relative content of constituents from H. longipes roots.
Inhibitory Activity of Heliopsis longipes Extract on Fusarium oxysporum f. sp lycopersici
Susana González Morales, María Liliana Flores López, Adalberto Benavides Mendoza, Alberto Flores Olivas
Abstract
A total of 17 Fusarium sp. isolates were obtained from tomato roots with wilt symptoms. Nine isolates were identified as Fusarium oxysporum f. sp. lycopersici; isolates pathogenicity was determined. More severe symptoms were caused by one of the isolates named FOL10. Such isolate was selected to have the Heliopsis longipes ethanol extract minimum inhibitory dose evaluated in vitro. The affinin concentration was determined as the extract main component. The estimated median lethal dose was 164.2 μg -1 -1 mL of affinin, and the lethal dose 90 was 348.6 μg mL of affinin.
Affinin (Spilanthol), Isolated from Heliopsis longipes, Induces Vasodilation via Activation of Gasotransmitters and Prostacyclin Signaling Pathways
Jesús Eduardo Castro-Ruiz, Alejandra Rojas-Molina, Francisco J. Luna-Vázquez, Fausto Rivero-Cruz, Teresa García-Gasca and César Ibarra-Alvarado
Abstract
Heliopsis longipes roots have been widely used in Mexican traditional medicine to relieve pain, mainly, toothaches. Previous studies have shown that affinin, the major alkamide of these roots, induces potent antinociceptive and anti-inflammatory activities. However, the effect of H. longipes root extracts and affinin on the cardiovascular system have not been investigated so far. In the present study, we demonstrated that the dichloromethane and ethanolic extracts of H. longipes roots, and affinin, isolated from these roots, produce a concentration-dependent vasodilation of rat aorta. Affinin-induced vasorelaxation was partly dependent on the presence of endothelium and was significantly blocked in the presence of inhibitors of NO, H2S, and CO synthesis (NG-nitro-L-arginine methyl ester (L-NAME), DL-propargylglycine (PAG), and chromium mesoporphyrin (CrMP), respectively); K+ channel blockers (glibenclamide (Gli) and tetraethyl ammonium (TEA)), and guanylate cyclase and cyclooxygenase inhibitors (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and indomethacin (INDO), respectively). Our results demonstrate, for the first time, that affinin induces vasodilation by mechanisms that involve gasotransmitters, and prostacyclin signaling pathways. These findings indicate that this natural alkamide has therapeutic potential in the treatment of cardiovascular diseases.
Antimutagenic properties of affinin isolated from Heliopsis longipes extract
Myriam Arriaga-Alba, María Yolanda Rios and Myrna Déciga-Campos
Abstract
Heliopsis longipes (A. Gray) Blake, known as “chilcuage” or “chilcuan” in Mexico, is traditionally utilized for its analgesic and anesthetic properties. The major metabolite, affinin, alongside the plant’s ethanol extract, has demonstrated antinociceptive effects in mice and produces a mix of antioxidant chlorophylls, polyamines, and potential antimutagens. The current study explored the use of affinin, extracted from H. longipes, as an antimutagenic and possibly anticarcinogenic agent, employing the Ames assay to evaluate its mutagenic properties against various mutagens in Salmonella typhimurium strains TA98, TA100, and TA102 with and without S9 metabolic activation. Results indicated that the H. longipes extract and affinin were not toxic, with affinin significantly reducing frameshift mutations induced by 2-aminoanthracene and oxidative DNA damage caused by norfloxacin by up to 50%. These findings suggest that affinin is not only non-mutagenic but also possesses significant antimutagenic properties, reinforcing the plant’s traditional use in treating pain and potentially offering benefits related to its antiradical activity.
Antinociceptive Effect of Heliopsis longipes Extract and Affinin in Mice
Wolfgang Barz, Rudolf Bauer, Veronika Butterweck, João Batista Calixto, Thomas Efferth, Jerzy W. Jaroszewski, Ikhlas Khan, Wolfgang Kreis, Irmgard Merfort, Kurt Schmidt, Thomas Simmet, Hermann Stuppner, Yang-Chang Wu, Yang Ye
Abstract
Heliopsis longipes is used as analgesic in Mexican traditional medicine. The present study assesses the possible antinociceptive effect of Heliopsis longipes and describes the pharmacological mechanisms of action of the antinociceptive effect of affinin, identified as the one active principle in Heliopsis longipes acetone extract. Intraperitoneal administration of H. Longipes extract and affinin produced a dose-dependent antinoceptive effect when assessed in mice submitted to acetic acid and capsaicin tests. Affinin-induced antinociception (30 mg/kg, i.p.) was blocked by naltrexone (1 mg/kg, s.c.), p-chlorophenylalanine (5 mg/kg s.c.) suggesting that its pharmacological effect could be due to the activation of opiodergic, serotoninergic and GABAergic systems. In addition, the antinociceptive effect of affinin was attenuated by pretreatment with 1H-oxadiazolo quinoxalin-1-one (1 mg/kg, s.c.) suggesting that the nitric oxide-k+ channels pathway could be involved in its mechanism of action. These results suggest that affinin itself or its derivatives may have potential antinociceptive effects.
Proangiogenic Effect of Affinin and an Ethanolic Extract from Heliopsis longipes Roots: Ex Vivo and In Vivo Evidence
Paola Estefanía García-Badillo, Anaguiven Avalos-Soriano, Josué López-Martínez, Teresa García-Gasca and Jesús Eduardo Castro-Ruiz
Abstract
Angiogenesis, the formation of new blood vessels, underlies tissue development and repair. Some medicinal plant-derived compounds can modulate the angiogenic response. Heliopsis longipes, a Mexican medicinal plant, is widely used because of its effects on pain and inflammation. The main bioactive phytochemicals from H. longipes roots are alkamides, where affinin is the most abundant. Scientific studies show various medical effects of organic extracts of H. longipes roots and affinin that share some molecular pathways with the angiogenesis process, with the vasodilation mechanism of action being the most recent. This study investigates whether pure affinin and the ethanolic extract from Heliopsis longipes roots (HLEE) promote angiogenesis. Using the aortic ring rat assay (ex vivo method) and the direct in vivo angiogenesis assay, where angioreactors were implanted in CD1 female mice, showed that affinin and the HLEE increased vascular growth in a dose-dependent manner in both bioassays. This is the first study showing the proangiogenic effect of H. longipes. Further studies should focus on the mechanism of action and its possible therapeutic use in diseases characterized by insufficient angiogenesis.
Expanding description and taxonomic aspects of Heliopsis longipes (Asteraceae: Heliantheae)
Virginia Gabriela Cilia-López, Juan Antonio Reyes-Agüero, Juan Rogelio Aguirre-Rivera, Bertha Irene Juárez-Flores
Abstract
Heliopsis longipes is, economically, the most important species of its genus, because its root has several traditional uses in Mexico. However, there are still unknown aspects of their morphology and biology. The objectives of this study were: a) to expand the botanical description of the species, b) to discuss the differences of H. longipes with some congeners, c) to document their phenology. Heliopsis longipes has an ascending to decumbent habit, ovate-oblong leaves and the stem is relatively long and it is microendemic in Sierra Alvarez and Sierra Gorda. Morphologically, it has more affinities with H. procumbens. The reproduction of H. longipes occurs during the wet season of the year
Distribution of Heliopsis longipes (Heliantheae: Asteraceae), an endemic resource from Central-Mexico
Cilia-López, V. Gabriela; Aguirre-Rivera, J. Rogelio; Espinosa-Reyes, Guillermo; Flores-Cano, Jorge A.; Reyes-Agüero, J. Antonio; Juárez-Flores, Bertha I.
Abstract
Heliopsis longipes is a perennial herb endemic to Sierra de Álvarez and Sierra Gorda in central México. It is a species of major economic importance within its genus, due to the traditional uses of its root. Chemical and pharmacological studies have been performed with H. longipes roots, however recent studies regarding its distribution are lacking. The objective of the current study was to establish and document the geographic distribution and existing ecologic conditions of H. longipes. A review of published works regarding this species, as well as an examination of herbarium specimens has been conducted. Explorations in Sierra de Álvarez and Sierra Gorda were also conducted to locate populations of H. longipes. In each site, the physical and biological factors associated with the population were recorded. Heliopsis. longipes is distributed in the states of San Luis Potosí, Guanajuato and Querétaro, in steep canyons with oak and oak-pine forests and substrates of igneous origin, in Sierra de Álvarez and Sierra Gorda.
Determination of the Insecticidal Activity of Heliopsis longipes A. Gray Blake, an Endemic Plant of the State of Guanajuato
Alejandro Hern.ndez-Morales; Jackeline Lizzeta Arvizu-G.mez; Blanca Estela G.mez-Luna; Enrique Ram.rez-Ch.vez; Mar.a del Rosario Abraham-Ju.rez; Gerardo Mart.nez-Soto and Jorge Molina-Torres
Abstract
Mosquitoes are involved in transmission of infectious diseases like malaria which agect human health, causing economic losses due to expensive treatments and job incapacity of patients. Strategies to minimize transmission of this disease are the employ of chemical insecticides that are excellent methods to reduce insect populations; however it causes deleterious egects on human health and environmental damage. Therefore is necessary to explore harmless alternatives, such as plant extracts which are potential source of natural insecticides. In this work we evaluated insecticidal properties of Heliopsis longipes A. Gray Blake against third instar larvae of Anopheles albimanus, malaria vector. Results showed that H. longipes A. Gray Blake has insecticide properties to control insect involved in malaria transmission.
Key words: Heliopsis longipes A. Gray Blake, Alcamides, Natural insecticides.
The genus Heliopsis (Heliantheae; Asteraceae) in Mexico and the alkamides present in its roots.
Abraham García-Chávez, Enrique Ramírez Chávez and Jorge Molina-Torres
Abstract
The genus Heliopsis belongs to the tribe Heliantheae in the Asteraceae family. It includes 14 species, most of them endemic to Mexico. One of the species, Heliopsis longipes, is utilized in traditional medicine. The high level of alkamides in its roots has already been chemically characterized. The study of these metabolites is displayed in a comparative analysis on the presence of alkamides in six Mexican species of Heliopsis. The quantitative and qualitative analysis of the alkamides stored in roots of each species is presented. Chemotaxonomic importance of alkamides in this genus is also discussed. In this work we review and update the bibliography referring to the taxonomy of the genus.
Key words: alkamides, Asteraceae, Heliantheae, Heliopsis, Mexico.
Ethnobotany of Heliopsis Longipes (Asteraceae Heliantheae)
V. Gabriela Cilia-L.Pez, J. Rogelio Aguirre-Rivera, Juan Antonio Reyes-Ag.Ero and Bertha I. Ju.Rez-Flores
Abstract
Heliopsis longipes known as chilcuague is a perennial herb endemic to Sierra de Álvarez and Sierra Gorda, where the States of San Luis Potos., Guanajuato and Quer.taro converge, and its root has traditional uses. However, there is a lack of studies that document the current traditional uses within its entire distribution area. The objectives of this research were to determine the traditional forms of use of chilcuague, and to determine the current demand of its root. Gathers, merchants, and consumers in traditional markets (tianguis) in San Luis Potos., Guanajuato and Quer.taro were interviewed. The root of chilcuague is economically and culturally important. It has diverse traditional uses and its sale is practiced all yearlong at the traditional markets of San Luis de la Paz, Dr. Mora, and San Jos. Iturbide, Guanajuato; Rioverde and San Ciro, San Luis Potos.; and Jalpan, Quer.taro. Chilcuague is used against parasites by adding roots over food. Moreover, a correlation with age of informants and knowledge of the uses of the plant was found. A higher amount of dried roots of chilcuague were sold in places with more traditional uses.
Key words: chilcuague, parasites, traditional uses, Sierra Gorda