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Antioxidant activity of enteral formulas
No full textUloga prehrane u prevenciji i liječenju brojnih kroničnih nezaraznih bolesti sve se više prepoznaje
kao ključna za održavanje zdravlja odnosno postizanje optimalnih ishoda liječenja. U tom kontekstu
se uz klasičnu dijetoterapiju sve više primjenjuje i ciljana vitaminsko-mineralna suplementacija, a
kod bolesti s izraženom malnutricijom, i enteralne formule optimiranog nutritivnog sastava. Osim
izbalansiranog sastava esencijalnih makro- i mikronutrijenata nove generacije enteralnih formulacija
vrlo često sadrže i neesencijalne sastavnice dokazanih bioloških učinaka, najčešće iz skupina spojeva
s antioksidativnim učinkom. U ovom radu istražen je učinak modifikacije standardne enteralne
formulacije dodatkom funkcionalnih sastavnica s obzirom na sadržaj polifenola i drugih spojeva s
antioksidacijskom aktivnošću. Ukupni sadržaj polifenola određen je Folin-Ciocalteu metodom
nakon odmašćivanja i uklanjanja interferirajućih proteina Carez reagensom. Antioksidacijski
potencijal određen je spektrofotometrijski u istim uzorcima, TEAC metodom. Dobiveni rezultati
pokazali su da se dodatkom resveratrola u formulaciju, kao i zamjenom dijela sirovina cjelovitim
žitaricama značajno povećava antioksidativna aktivnost uzoraka te da se upravo polifenoli mogu
smatrati glavnim nositeljima antioksidativne aktivnosti analiziranih proizvoda. Utvrđeno je da se
skladištenjem analiziranih proizvoda na sobnoj temperaturi ukupni antioksidacijski potencijal malo,
ali statistički značajno smanjuje u uzorcima s dodatkom polifenola masline i resveratrola. Dobiveni
rezultati doprinijeti će razumijevanju trenutnih mogućnosti i ograničenja u razvoju inovativnih
formulacija za enteralnu primjenu.The role of nutrition in the prevention and treatment of numerous chronic indistinct diseases is
increasingly being recognized as key to maintaining health, i.e. achieving optimal treatment
outcomes. In this context, in addition to classical diet therapy, targeted vitamin and mineral
supplementation is increasingly used, and in diseases with severe malnutrition, enteral formulas with
an optimized nutritional composition. In addition to the balanced composition of essential macroand micronutrients, the new generation of enteral formulations very often contain non-essential
components with proven biological effects, most often from groups of compounds with antioxidant
effects. This paper investigated the effect of modifying the standard enteral formulation with the
addition of functional components with regard to the content of polyphenols and other compounds
with antioxidant activity. The total content of polyphenols was determined by the Folin-Ciocalteu
method in defatted samples, after removing interfering proteins with the Carez reagent. The
antioxidant potential was determined spectrophotometrically in the same samples, using the TEAC
method. The obtained results showed that the addition of resveratrol to the formulation, as well as
the replacement of part of the raw materials with whole grains, significantly increases the antioxidant
activity of the samples and that polyphenols can be considered the main carriers of the antioxidant
activity of the analyzed products. It was found that by storing the analyzed products at room
temperature, the total antioxidant potential decreases slightly, but statistically significantly in
samples enriched with olive-polyphenols and resveratrol. The obtained results will contribute to the
understanding of current opportunities and limitations in the development of innovative
formulations for enteral administration
Analysis of mRNA BNT162B vaccine effect on angiogenic factors PlGF and sFlt-1
No full textPojava tromboembolijskih incidenata s početkom procjepljivanja protiv SARS-CoV-2 zasigurno je jedna od najvećih dilema koju je donijela pandemija Covid-19. Vodeći se činjenicom da cijepljenje oponaša prirodnu infekciju, pretpostavljeno je da uslijed poremećene regulacije renin-angiotenzin sustava vezanjem šiljastog proteina može doći do angiogene neravnoteže te hiperkolagulabilnog stanja koje je u pozadini tromboemboliskih incidenata. U skladu s tim, ovim istraživanjem ispitivan je učinak mRNA BNT162b cjepiva na koncentracije angiogenih čimbenika PlGF i sFlt-1. Konkretni ciljevi bili su ispitati utjecaj cijepljenja na koncentracije PlGF i sFlt-1, razliku u koncentracijama između podskupina ispitanika bez hipertenzije i s hipertenzijom, te povezanost PlGF i sFlt-1 s dobi te indeksom tjelesne mase u pojedinim vremenskim točkama nakon cijepljenja. Predmet ovog istraživanja bila su 33 odrasla ispitanika cijepljena mRNA BNT162b cjepivom. Koncentracije čimbenika mjerene su istovjetno u 5 vremenskih točaka: T0 – točka mjerenja neposredno prije 1. doze (unutar 24h); T1 – točka mjerenja 7 dana nakon 1. doze; T2 – točka mjerenja 14 dana nakon 1. doze; T3 – točka mjerenja 7 dana nakon 2. doze; T4 – točka mjerenja 14 dana nakon 2. doze. Za laboratorijsku analizu je korištena elektrokemiluminiscentna imunokemijska metoda (ECLIA) temeljena na sendvič principu. Statistički značajna razlika između određivanih koncentracija kod svh ispitanika procijenjena je Wilcoxon signed-rank testom, dok je statistički značajna razlika između određivanja kod ispitanika bez hipertenzije i s hipertenzijom ispitana Mann-Whitneyevim testom. Moguća povezanost čimbenika s dobi te indeksom tjelesne mase ispitana je Spearmanovom korelacijom. Detaljnom analizom, unatoč određenim statistički značajnim razlikama, nisu pronađene klinički značajne promjene u koncentracijama PlGF i sFlt-1 uvjetovane cijepljenjem, niti je utvrđena razlika u koncentracijama između podskupina ispitanika bez hipertenzije i s hipertenzijom. Također, nije utvrđena značajna povezanost koncentracija PlGF i sFlt-1 u pojedinim točkama nakon cijepljenja s dobi, niti s indeksom tjelesne mase. S obzirom na dobivene rezultate, nije dokazano da pretpostavljeni mehanizam ima ulogu u nastanku tromboembolijskih incidenata nakon cijepljenja mRNA BNT162b cjepivom.The incidence of thromboembolic events after SARS-CoV-2 vaccination is still one of the biggest concerns of the Covid-19 pandemic. Since vaccination imitates natural infection, we hypothesized that the dysregulation of the renin-angiotensin system, caused by spike protein binding, could induce an angiogenic imbalance leading to a hypercoagulable state and thromboembolic events. Based on this hypothesis, we tested dynamics in angiogenic factor concentration changes caused by the mRNA BNT162b vaccine. The specific aims of this study were to test the effect of vaccination on PlGF and sFlt-1 concentrations, the concentration difference between subgroups without and with hypertension, and to examine the correlation between PlGF and sFlt-1 with age and body mass index at defined time points after vaccination. The study included 33 adult subjects who were vaccinated with the mRNA BNT162b vaccine. Factors' concentrations were measured at five time points: T0 – measurement point before the1st dose (within 24h); T1 – measurement point 7 days after the 1st dose; T2 – measurement point 14 days after the 1st dose; T3 – measurement point 7 days after the 2nd dose; T4 – measurement point 14 days after the 2nd dose. Factors were measured using electrochemiluminescence method (ECLIA) based on the sandwich principle. The statistically significant difference between the measurements for all subjects was tested using Wilcoxon signed-rank test, while the statistically significant difference between subjects without and with hypertension was tested by using Mann-Whitney test. The possible correlation was tested by Spearman's correlation. Although statistically significant differences were obtained, they are indicated to be clinically irrelevant. No clinically significant changes in PlGF and sFlt-1 concentrations were found after vaccination. There were no differences in concentrations between subjects without hypertension and those with hypertension. No correlation was found between PlGF and sFlt-1 concentrations with age or body mass index at any time point after vaccination. According to the results, it has not been proven that the assumed mechanism participates in the incidence of thromboembolic incidents after vaccination with the mRNA BNT162b vaccine
Chemical characterization and biological effects of polyphenols in wines
No full textVina sadrže različite biološki aktivne polifenolne spojeve za koje je utvrđeno da imaju brojne pozitivne učinke na zdravlje.
Polifenolni profil vina često se koristi za procjenu njihove kvalitete i autentičnosti. U okviru doktorskog rada provedena je
usporedna kemijska karakterizacija polifenola u dvadeset i pet komercijalnih crnih i bijelih vina proizvedenih u različitim
hrvatskim vinogradarskim regijama od osam crnih i devet bijelih sorti grožđa. U tu je svrhu razvijena i validirana metoda
visokoučinske tekućinske kromatografije (HPLC-DAD) s direktnim injektiranjem vina. Analizirane su dvadeset i četiri
polifenolne sastavnice vina te razvrstane u pet skupina na temelju njihove strukture: fenolne kiseline, flavan-3-ole, antocijane,
flavonoide i stilbene. Crna vina sadržavala su značajno veće koncentracije polifenola od bijelih vina te je kod njih uočena
velika varijabilnost u sastavu. Specifičnim polifenolnim sastavom i sadržajem posebice su se istaknula vina proizvedena od
autohtonih hrvatskih sorti plavac mali i babić. Primjenom multivarijatne analize ustanovljene su sličnosti i razlike u
polifenolnim profilima hrvatskih monosortnih vina te detektirani važni diferencijatori. Glavni predstavnici polifenolnih
sastavnica koji su bili značajno zastupljeni u istraživanim vinima pokazali su snažno antioksidacijsko djelovanje te sposobnost
inhibicije aktivnosti enzima Src-tirozin-kinaze, α-glukozidaze i acetilkolinesteraze. Provedeno istraživanje pružilo je nove
spoznaje o polifenolnim profilima hrvatskih vina, dajući doprinos njihovoj sortnoj diferencijaciji, autentifikaciji i kontroli
kvalitete. Ustanovljeno je da su analizirana crna vina, prvenstveno ona dobivena iz hrvatskih autohtonih crnih sorti grožđa,
vrijedan izvor polifenola sa snažnim antioksidacijskim svojstvima i sposobnošću inhibicije enzima koji predstavljaju važne
terapijske mete u prevenciji i liječenju nekih kroničnih bolesti povezanih sa starenjem.Wines contain various biologically active polyphenolic compounds that are belived to have numerous beneficial effects on
human health. The polyphenolic profile of wines is often used to evaluate their quality and authenticity. In this doctoral thesis, a comparative chemical characterization of polyphenols was performed in twenty-five commercial red and white wines produced in different Croatian wine-growing regions from eight black and nine white grape varieties. For this purpose, a method of high-performance liquid chromatography (HPLC-DAD) with direct injection of wine was developed and validated. Twentyfour polyphenolic components of the wines were studied and classified into five groups based on their structure: phenolic acids, flavan-3-ols, anthocyanins, flavonoids and stilbenes. Black wines contained significantly higher concentrations of polyphenols than white wines, and a great variability in their composition was found. Wines produced from the native Croatian varieties Plavac mali and Babić were characterized by their specific polyphenolic composition and content. Multivariate analysis was used to determine similarities and differences in the polyphenolic profiles of Croatian monovarietal wines and to identify important differentiators. The main representatives of polyphenolic components, which were significantly present in the studied wines, showed strong antioxidant activity and the ability to inhibit the activity of Src tyrosine kinase, α-glucosidase and acetylcholinesterase enzymes. The conducted research provided new insights into the polyphenolic profiles of Croatian wines, thus contributing to their varietal differentiation, authentication and quality control. It was found that the analyzed red wines, especially those obtained from Croatian autochthonous red grape varieties, are a valuable source of polyphenols with strong antioxidant properties and the ability to inhibit enzymes that are important therapeutic targets in the prevention and treatment of some chronic diseases associated with aging
Coenzyme Q10 in the fight against signs of aging caused by oxidative stress
No full textStarenje kože složen je proces koji ovisi o različitim unutarnjim i vanjskim čimbenicima te uključuje brojne mehanizme na staničnoj i molekularnoj razini. Povezan je s mitohondrijskom disfunkcijom, povećanim stvaranjem slobodnih radikala te gubitkom stanične energije, što dovodi do vidljivih znakova starenja poput bora, gubitka elastičnosti i suhoće kože. Zbog antioksidativnih i protektivnih svojstava, ali i spoznaje da se mogućnost organizma da ga proizvede smanjuje s godinama, u posljednjem se desetljeću značajno povećala dostupnost proizvoda za njegu kože i dodataka prehrani koji sadrže koenzim Q10. Kao endogena, lipofilna molekula ima ključnu ulogu u mitohondrijskom lancu prijenosa elektrona te djeluje kao snažan antioksidans, neutralizirajući slobodne radikale i tako štiteći stanične strukture od oksidativnog oštećenja. Upravo su intrinzično (genetski uvjetovano) i ekstrinzično (UV zračenjem i okolišem inducirano) starenje kože područja u kojima koenzim Q10 pokazuje terapijski potencijal. Brojna su istraživanja pokazala da lokalna primjena Q10 unutar brojnih formulacija može poboljšati izgled kože smanjenjem dubine bora, poboljšanjem elastičnosti te stimulacijom regenerativnih procesa. S druge strane, oralna suplementacija pokazuje bolje rezultate kada se koristi u kombinaciji s drugim aktivnim tvarima poput kolagena i ostalih antioksidansa.Skin aging is a complex process influenced by various intrinsic and extrinsic factors and involves numerous mechanisms at the cellular and molecular levels. It is associated with mitochondrial dysfunction, increased production of free radicals, and a decline in cellular energy, which lead to visible signs of aging such as wrinkles, loss of elasticity, and skin dryness. Due to its antioxidant and protective properties, as well as the fact that the body's ability to synthesize it decreases with age, the availability of skincare products and dietary supplements containing coenzyme Q10 has significantly increased over the past decade. As an endogenous, lipophilic molecule, CoQ10 plays a key role in the mitochondrial electron transport chain and acts as a potent antioxidant by neutralizing free radicals and thus protecting cellular structures from oxidative damage. Both intrinsic (genetically determined) and extrinsic (UV radiation- and environment-induced) skin aging are areas where CoQ10 shows therapeutic potential. Numerous studies have demonstrated that topical application of CoQ10 in various formulations can improve skin appearance by reducing wrinkle depth, enhancing elasticity, and stimulating regenerative processes. On the other hand, oral supplementation has shown better results when used in combination with other active compounds such as collagen and other antioxidants
The reliability of lipemia interference removal in routine coagulation tests through high-speed centrigufation
No full textLipemija je jedna od najčešćih optičkih interferencija koja se nerijetko javlja kod bolesnika na parenteralnoj prehrani i predstavlja izazov za pravilnu analizu koagulacijskih pretraga. Parenteralna prehrana podrazumijeva direktan unos nutrijenata u krv pacijenta, uključujući i lipoproteinske čestice, od kojih su za uzrokovanje lipemije najzasluţniji hilomikroni, srednje i velike VLDL čestice. Koagulometrijske metode koriste se za rutinsko određivanje kogulacijskih parametara, PV-a, APTV-a i fibrinogena, a temelje se na nastanku fibrinskog ugruška, koji se detektira optičkim metodama. Lipemija moţe uzrokovati laţne rezultate ili ometati analizu koagulacijskih parametara koji se određuju foto-optičkom detekcijom. Takav način detekcije koristi uređaj Sysmex CS-5100, koji je korišten u sklopu ovog istraţivanja za određivanje PV-a, APTV-a i fibrinogena. U istraţivanju je korišteno 60 uzoraka iz rutinske analize KBC-a Zagreb, u kojim je lipemija izazvana in vitro pomoću SMOFlipid emulzije, a eliminirana metodom centrifugiranja velike brzine. Dobiveni rezultati pokazuju statistički značajna odstupanja u skupinama s prosječnim indeksom lipemije od 1600 mg/dL, uz iznimke koje su rezultat predanalitičkih čimbenika ili osjetljivosti korištenog Wilcoxonovog statističkog testa. Značajne apsolutne razlike između lipemičnih i nelipemičnih uzoraka za pretrage PV(omjer), PV-INR i APTV uočene su iznad graničnog indeksa lipemije od 800 mg/dL, dok je učinkovitost metode eliminacije za pretragu fibrinogen potvrđena do indeksa lipemije od 1700 mg/dL. Centrifugiranje velike brzine pokazalo se kao učinkovita metoda eliminacije lipemije do određenog stupnja lipemije, što moţe pomoći u dobivanju pouzdanijih rezultata rutinskih koagulacijskih pretraga.Lipemia is one of the most common optical interferences that frequently occurs in patients receiving parenteral nutrition and presents a challenge for the accurate analysis of coagulation tests. Parenteral nutrition involves the direct infusion of nutrients into the patient’s bloodstream, including lipoprotein particles, with chylomicrons, medium, and large VLDL particles being primarily responsible for causing lipemia. Coagulation methods are used for routine determination of coagulation parameters, such as PT, APTT, and fibrinogen, and are based on the formation of a fibrin clot, which is detected by optical methods. Lipemia can cause false results or interfere with the analysis of coagulation parameters that are determined using photo-optical detection. This detection method uses the Sysmex CS-5100 device, which was employed in this study to determine PT, APTT, and fibrinogen. The study utilized 60 samples from routine analysis at the University Hospital Center Zagreb, in which lipemia was induced in vitro using SMOFlipid emulsion, and was eliminated by high-speed centrifugation. The results show statistically significant deviations in the groups with an average lipemia index of 1600 mg/dL, with exceptions due to pre-analytical factors or the sensitivity of the Wilcoxon statistical test used. Significant absolute differences between lipemic and non-lipemic samples for PT (ratio), PT-INR, and APTT were observed above a lipemia index threshold of 800 mg/dL, while the effectiveness of the elimination method for fibrinogen testing was confirmed up to a lipemia index of 1700 mg/dL. High-speed centrifugation proved to be an effective method for eliminating lipemia to a certain degree, which may help obtain more reliable results in routine coagulation tests
NMR study of the effect of UV light on the chlorination reaction of ifosfamide
No full textIfosfamid se nalazi u grupi citotoksičnih lijekova kojeg je JRC Europske komisije svrstala u
zabrinjavajuće spojeve obzirom da još uvijek nije razvijena učinkovita metoda njegovog uklanjanja
iz otpadnih voda. Najvećim dijelom se izlučuje urinom nepromijenjen i kao takav dolazi u sustav
otpadnih voda. U ovom radu je proučavana reakcija kloriranja ifosfamida u tami i pod utjecajem
različitih tipova elektromagnetskog zračenja (bijelo svjetlo, UV svjetlo), s ciljem utvrđivanja
učinkovitosti najčešće korištenih metoda obrade otpadnih voda u uklanjanju navedenog onečišćivala
iz otpadnih voda. Identificiran je glavni produkt reakcije kloriranja i razmotrena njegova stabilnost
u mraku i pod svjetlom, kako bi se utvrdila njegova relevantnost za ekotoksikološka ispitivanja. Iz
dobivenih 1H, 13C, 31P NMR spektara, može se zaključiti da dolazi do kloriranja egzocikličkog
dušika iz N-(2-kloroetil) skupine ifosfamida. Klorirani produkt je fotolabilan u prisutnosti UV
svjetla i njegova razgradnja je jako ovisna o intenzitetu zračenja, dok je sam ifosfamid stabilan.
Obzirom da se pregradnja kloriranog ifosfamida u ifosfamid vjerojatno odvija putem homolitičkog
cijepanja, ali i zbog same toksičnosti ifosfamida, potrebno je provesti daljnja istraživanja
ekotoksičnosti kloriranih produkata.Ifosfamide is in the group of cytotoxic drugs classified by the JRC of the European Commission as
a compound of concern, since an effective method of its removal from wastewater has not yet been
developed. It is mostly excreted in the urine unchanged and as such enters the wastewater system.
In this paper, the chlorination reaction of ifosfamide in the dark and under the influence of different
types of electromagnetic radiation (white light, UV light) was studied, with the aim of determining
the effectiveness of the most commonly used wastewater treatment methods in removing this
pollutant from wastewater. The main product of the chlorination reaction was identified and its
stability in the dark and under light was considered, in order to determine its relevance for
ecotoxicological studies. From the obtained 1H, 13C, 31P NMR spectra, it can be concluded that
chlorination of exocyclic nitrogen from the N-(2-chloroethyl) group of ifosfamide occurs. The
chlorinated product is photolabile in the presence of UV light and its degradation is highly dependent
on the intensity of radiation, while ifosfamide itself is stable. Given that the conversion of
chlorinated ifosfamide to ifosfamide probably occurs by homolytic cleavage, but also due to the
toxicity of ifosfamide itself, further research on the ecotoxicity of chlorinated products needs to be
conducted
Production optimisation and biological activity of glycerol extracts of medicinal plants for application in dermatopharmacy
No full textRazvijene su zelene metode ultrazvučne ekstrakcije bioaktivnih sastavnica biljnih droga
Berberidis radicis cortex, Echinaceae purpureae herba, Liquiritiae radix i Silybi mariani fructus
pomoću mješavina glicerola i vode. Za optimizaciju ekstrakcija korištena je metodologija
odziva površine prema Box-Behnkenovom dizajnu. Ispitan je utjecaj većeg broja neovisnih
varijabli, kao što su udio glicerola u ekstrakcijskoj smjesi, temperatura, snaga ultrazvuka,
vrijeme ekstrakcije, dodatak askorbinske kiseline u reakcijsku smjesu te omjer droga/otapalo,
na ishode ekstrakcije. Ovisne varijable bile su koncentracija odabranih bioaktivnih sastavnica
te antiradikalna aktivnost ekstrakata. Na ishode UAE-a najviše su utjecali udio glicerola i
vrijeme ekstrakcije te temperatura i snaga ultrazvuka. Na osnovi eksperimentalnih podataka
izrađeni su ekstrakcijski modeli, a njihova prikladnost potvrđena je visokom podudarnošću
eksperimentalno određenih vrijednosti ovisnih varijabli s onima dobivenim teorijskim
modelima. Priređenim ekstraktima ispitan je antioksidativni učinak te učinak na enzime i
procese koji utječu na izgled i zdravlje kože. Glicerolni ekstrakti odabranih biljnih droga
djelovali su kao antioksidansi, te su učinkovito inhibirali enzime tirozinazu, kolagenazu,
elastazu, hijaluronidazu i lipoksigenazu, kao i koagulaciju proteina. Posebno istaknut bio je
inhibitorni učinak ekstrakata Liquiritiae radix na enzime elastazu i tirozinazu, koji je bio
usporediv ili jači od korištenih standarda dok su ekstrakti droge Echinaceae purpureae herba
snažno inhibirali hijaluronidazu. Ekstrakti Echinaceae purpureae herba bili su biokompatibilni
s HaCaT staničnim kulturama te su potaknuli proces cijeljenja rana na in vitro modelu. Glicerol,
u koncentracijama korištenim u ekstraktima, inhibirao je toplinom induciranu koagulaciju
proteina aktivno pridonoseći učinku ekstrakata. Sve navedeno čini glicerolne ekstrakte
odabranog ljekovitog bilja potencijalnim aktivnim sastavnicama u dermatofarmaceutskim
proizvodima.Introduction
Over time, due to the action of internal and external adverse factors, a number of
structural changes occur in the skin. Those changes include changes in pigmentation, elasticity
or hydration, which all manifest themselves as skin aging. To combat those changes, numerous
dermatopharmaceutical products are being developed with the aim of displaying positive effects
on the skin’s health and appearance. The bioactive components of such products are often
secondary metabolites of medicinal plants, such as polyphenols and alkaloids. They are known
for their beneficial effect on the skin including antioxidant, anti-inflammatory, and antihyperpigmentation
effects.
Besides their skin-related beneficial effects, contemporary cosmetic and dermatological
products are often prepared so that they meet the ethical and environmental demands for a
sustainable production. One of the pivotal aspects of producing eco-friendly plant extracts is
selection of eco-friendly extraction techniques and solvents, and ultrasound-assisted extraction
is one of the most used green extraction techniques. This technique achieves high yields with
lower energy and solvent use by applying sound waves to break cell walls, expediting
compound release. Furthermore, green solvents, characterized by their biodegradability, nontoxicity
and low flammability are an integral part of green extraction methods. Glycerol, a nontoxic
and biodegradable solvent of natural origin, is characterized by all the characteristics that
a green solvent should display. It also has an additional advantage which makes it highly
suitable for cosmetic applications due to its influence on viscosity and stability of the cosmetic
product and the moisture-retaining properties on the skin. Therefore, glycerol offers a valuable
eco-friendly option for plant-based extract production.
Numerous scientific studies show that herbal drugs Berberidis radicis cortex,
Echinaceae purpureae herba, Liquiritiae radix, and Silybi mariani fructus have a significant
dermatopharmaceutical potential. Therefore, in this dissertation, the optimization of glycerolbased
ultrasound-assisted extraction of the phenolic and other active components from these
drugs was performed using chemometric methods. The antioxidant activity of optimized
extracts was determined and their ability to inhibit the enzymes collagenase, elastase, tyrosinase
and lipoxygenase was examined. Additionally, their effect on hyaluronidase, biocompatibility
with HaCaT cells as well as their effect on wound healing in the "scratch" test was assessed.
Methods
The identity of the herbal drugs was confirmed by the procedures described in the
relevant pharmacopoeial monographs and other appropriate scientific literature. Prior to the
extraction, plant material was pulverized and sieved to obtain uniform particle size. In the
preliminary extractions, comparison of macerations using either glycerol/water or
ethanol/water mixtures was performed in order to select the optimal solvent. In addition to that,
the effectiveness of ultrasound assisted extraction with glycerol/water mixtures was compared
with the effectiveness of maceration performed using the same solvent. In order to obtain
extracts with desired chemical and biological characteristics ultrasound-assisted extraction was
optimized using response surface methodology. Box-Behnken design and two-level factorial
design were used for design of these experiments. Influences of several factors (independent
variables; glycerol concentration, extraction duration, ultrasound power, temperature, addition
of ascorbic acid to the extraction mixtures and the weight of herbal drug used for the extraction)
on the extraction outcomes (dependent variables) were assessed.
Concentrations of the selected bioactive ingredients in the extracts, berberine from
Berberidis radices cortex, cichoric and caftaric acid from Echinaceae purpureae herba, glabridin
and isoliquiritigenin from Liquiritiae radix, and silymarin (flavonolignans silibinin A and B,
isosilibinin A and B, silicristin and silidianin concentrations) from Silybi mariani fructus, were
determined by high-performance liquid chromatography. UV-VIS spectrophotometry was used
for determination of total phenolic compounds in Liquiritiae radix. Total phenolic acids in
Echinaceae purpureae herba was calculated as the sum of cichoric and caftaric acid
concentrations. Relative extraction efficiency of total phenols from Liquiritiae radix was
calculated as the ratio of total phenol content and the weight of the plant material used for the
extraction.
Antioxidant potential of the extracts was determined using several methods. Antiradical
activity of the extracts was tested spectrophotometrically with 2,2-diphenyl-1-picrylhydrazyl
radical. The inhibition of oxidative degradation of unsaturated fatty acids was tested by
monitoring the kinetics of b-carotene degradation in the presence of linoleic acid at elevated
temperature, whereas the ability to chelate Fe2+ ions was determined in the reaction with
ferrozine. Butylated hydroxyanisole (BHA) and EDTA were used as positive controls,
respectively.
Appropriate spectroscopic methods were used to determine the ability of plant extracts,
as well as glycerol in the concentrations used in the extracts, to inhibit specific enzymes that
can affect the health and appearance of the skin. Tyrosinase inhibition was determined by
monitoring formation of dopaquinone from 3,4-dihydroxy-L-phenylalanine. Kojic acid was
used as a reference inhibitor. Elastase activity was tested with the substrate N-succinyl-Ala-
Ala-Ala-p-nitroanilide and oleanolic acid as a positive control. Collagenase activity was
determined by using N- 3-2-furyl-acryloyl-Leu-Gly-Pro-Ala as the substrate and gallic acid as
a positive control. Anti-hyaluronidase activity was performed in the assay with pdimethylaminobenzaldehide,
while tannic acid was used as positive control. The antiinflammatory
activity of plant extracts was tested by measuring the concentration of conjugated
diene resulting from the oxidation of linoleic acid by 5-lipoxygenase and in the reaction with
ovalbumin. Nordihydroguaiaretic acid and diclofenac were used as positive controls,
respectively. Evaluation of biocompatibility and wound healing effects were evaluated on
HaCaT cells, immortal keratinocyte cell line from human skin. Cell viability was assessed using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT test), while wound
healing effects were determined using the "scratch" test method.
Measurements were performed in triplicate and expressed as arithmetic mean ± standard
deviation. The activities in the performed assays were calculated by regression analysis and
expressed as an IC50 values. Statistical differences were examined using ANOVA and
corresponding post-hoc tests (Tukey's and Dunnett's post-hoc tests for comparison among the
extracts and the control, respectively). Validity of the response surface methodology models
was assessed using ANOVA.
Results
Preliminary experiments were focused on the comparison of the two extraction solvent
systems (glycerol/water mixtures vs. ethanol/water mixtures) and the two extraction techniques
(maceration vs. ultrasound assisted extraction). They were performed using the extraction of
phenolic acids from Echinaceae purpureae herba and silymarin from Silybi mariani fructus as
the models for further development of extraction procedures. Maceration results showed that
glycerol/water mixtures were equally effective solvents for the extraction of phenolic acids
from Echinaceae purpureae herba as ethanol/water mixtures. However, the efficiency of
glycerol/water for extraction of silymarin from Silybi mariani fructus was inferior to
ethanol/water mixtures. On the other hand, ultrasound-assisted extraction of target compounds
was superior to maceration in both cases. It achieved significantly higher yields of target
phenolics despite much shorter extraction time. In addition, the efficiency of glycerol/water for
ultrasound-assisted extraction of silymarin from Silybi mariani fructus reached the efficiency
of maceration using ethanol/water mixtures. Taking this into account, glycerolic ultrasoundassisted
extraction was proposed as the method of choice in further investigations performed
within this work.
Preliminary ultrasound-assisted extraction of Echinaceae purpureae herba, performed
according to the two-level factorial design, was used for initial selection of independent
variables for further extraction optimization. It was found that the extraction outcome was most
influenced by glycerol concentration, extraction time, temperature and ultrasound power. The
addition of ascorbic acid had a negative, time-dependent, influence on the concentration of all
the Echinaceae purpureae herba phenolic acids analyzed in this work.
Based on the results obtained in the preliminary experiments, further optimization of
the glycerol extraction of bioactive compounds from selected herbal drugs was performed.
Glycerol content and the temperature were used as independent variables in all the performed
extraction optimizations, while the other variables were selected on case-to-case basis.
Additional independent variable for optimization of berberine yield and antiradical activity in
Berberidis radicis cortex extraction was the ultrasonication power, while plant material to
solvent mass ratio was the additional variable for the extraction of total phenolic compounds,
glabridin and isoliquiritigenin from Liquiritiae radix. Extraction of Echinaceae purpureae herba
was optimized for the yield of caftaric acid, chicoric acid, total phenolic acids, and for
antiradical activity. Ultrasonication power and time were used as additional independent
variables, while time was the additional independent variable for preparation of Silybi mariani
fructus extracts optimized for the yield of silymarin and antiradical activity.
Further experiments were conducted based on the protocols established by Box-
Behnken design. Upon the preparation of the extracts and the determination of the target
dependent variables, the independent variables that significantly affected the extraction
efficiency were determined. The most important independent variables, the extraction
temperature and glycerol concentration, significantly influenced the efficiency in all the
performed experiments. In all the extractions conducted within this research, the extraction
outcome was proportional to the negative square value of the glycerol concentration. Also, a
negative linear effect of glycerol concentration was observed during the extraction of
Echinaceae purpureae herba, Berberidis radicis cortex and Liquiritiae radix. On the other hand,
the extraction temperature had a positive linear influence on the outcome of all the phenolic
compounds extractions performed within this work. Even though the other independent
variables were not employed in all the extractions performed herein, in individual cases they
also influenced extraction efficiency. For example, ultrasonication power influenced the
extraction of berberine from Berberidis radicis cortex, and the extraction of phenolic acids from
Echinaceae purpureae herba, while mass ratio of plant material to solvent influenced the
extraction of all the dependent variables from Liquiritiae radix. Extraction time significantly
influenced the content of target phenolic compounds from Echinaceae purpureae herba and
Silybi mariani fructus.
Based on the values of dependent variables in the prepared extracts (concentrations of
the target compounds and antiradical activity) response surface methodology was used to
propose the equations for extraction models and to calculate the values of independent variables
needed to achieve the extraction goals. Two extracts were prepared from Berberidis radicis
cortex. The extract optimized for berberine content contained 145.5 μg/mL of the alkaloid, and
it was prepared at 80 °C using 50% (w/w) glycerol and 144 W ultrasonication power. The extract
with maximum antiradical activity (IC50 = 58.88 μL extract/mL) was prepared at the same
temperature, but using lower glycerol content (30 %, w/w) and stronger ultrasonication (720
W). The conditions necessary for preparation of the Echinaceae purpureae herba extracts
optimized for caftaric acid, cichoric acid and total phenolic acid contents were essentially the
same. Therefore, only one extract was prepared using the calculated conditions: 70% glycerol
(w/w), temperature of 60 °C and 360 W of ultrasonication power. The extraction lasted for 60
min. Contents of caftaric acid, cichoric acid and total phenolic acid the extracts were 31.82
μg/mL, 113.11 μg/mL and 144.93 μg/mL, respectively. The Echinaceae purpureae herba extract
displaying the strongest antiradical activity was prepared at 60 °C and using 177 W
ultrasonication power. The extraction was performed with 50%, glycerol (w/w) and lasted for
55 min.
Liquiritiae radix extract optimized for total phenolics, obtained using 20% glycerol
(w/w), 70 °C and using 0.93 g of plant material, contained 854.6 μg/mL of the target compounds.
The extract with the highest relative extraction efficiency of total phenols contained 791.6
μg/mL of phenols per g of plant material. It was prepared using 30% glycerol (w/w), at 70 °C
and using 0.7 g of plant material. The conditions required for preparation of the extracts
optimized for glabridin and isoliquiritigenin contents coincided and only one extract was
prepared. The most appropriate extraction solvent for its preparation was 85% glycerol (w/w).
The extract was prepared at 70 °C using 1 g of plant material. Concentrations of glabridin and
isoliquiritigenin in thus prepared extract were 21.89 μg/mL and 6.23 μg/mL, respectively. The
Silybi mariani fructus extract with strongest antiradical activity (IC50 = 192.30 μL extract/mL)
was prepared at 60 °C, but using a rather low glycerol content (20%, w/w). The extraction was
performed for 60 min. The best conditions for the extraction of silymarin were 60 min at 80 °C
using 40% (w/w) glycerol. Silymarin content in the extract optimized for the highest
concentration of active substances, reached 99.6 μg/mL (expressed as silibinin).
The difference between the experimental results and the values calculated by the
theoretical models was less than 8% indicating the validity of the models. The prepared extracts
were used for further investigation of biological activities.
The optimized extracts displayed notable antioxidant activities in the performed
antioxidant assays. For example, Silybi mariani fructus extract optimized for antiradical activity
had a statistically equal effect to the employed positive control BHA, while the extract
optimized for silymarin content had a better effect than BHA in the test with b-carotene and
linoleic acid. Both Echinaceae purpureae herba extracts showed a statistically better effect than
BHA in the test with b-carotene. The Liquiritiae radix extract optimized for glabridin and
isoliquiritigenin content showed a better radical scavenging activity than BHA, while the
extract optimized for total phenolic content was as effective an iron chelator as EDTA.
The optimized extracts effectively inhibited enzymes and processes that negatively
affected the appearance and health of the skin, indicating the potential for positive effects on
inflammatory processes, pigmentation, skin firmness, hydration and elasticity. The activity of
several extracts in the performed assays was equal to or statistically higher than the activity of
standard inhibitors. By far the most effective among the tested optimized extracts were the
extracts prepared from Liquiritiae radix. The extract optimized for glabridin and
isoliqiuritigenin content was a stronger inhibitor of protein coagulation, as well as elastase and
tyrosinase activity than the employed positive controls diclofenac, ursolic and kojic acid.
Compared to the standard inhibitors, the extract optimized for total phenolic content was an
equally effective elastase inhibitor and a better tyrosinase inhibitor, while the extract optimized
for extraction efficiency of total phenols was an equally effective elastase and tyrosinase
inhibitor. Echinaceae purpureae herba extracts also displayed notable efficacy in the performed
assays. For example, the extract optimized for the highest content of phenolic acids and the
extract optimized for the most pronounced antiradical activity were more effective than tannic
acid in the hyaluronidase inhibition test, while the extract optimized for total phenolic acid
content was a statistically equally effective tyrosinase inhibitor as kojic acid. Glycerol, at the
concentrations in which it was present in the extracts, inhibited heat-induced protein
coagulation, actively contributing to the effect of the extracts. This ability of glycerol further
confirms that the advantages of using glycerol extracts in cosmetics go beyond its use as a green
solvent.
Biocompatibility and wound healing activity was investigated using Echinaceae
purpureae herba glycerol extracts. When tested for biocompatibility, neither the extracts nor
glycerol, did significantly adversely affect the viability of HaCaT cells in concentration of up
to 25 μL/mL. When used for in vitro investigation of wound healing using the in vitro "scratch"
model wound, the extracts used in these concentrations significantly increased wound healing
rate. After 48 h, the scratch surface in the cell monolayer treated with the extract optimized for
antiradical activity in concentration of 2.5 μL/mL reduced by approximately 60 %. Similar
results were observed in the extract optimized for phenolic acid content, indicating excellent
wound healing activity.
Conclusion
The performed research confirmed that glycerol is an effective solvent for the extraction
of the active components of herbal drugs: Berberidis radicis cortex, Echinaceae purpureae
herba, Liquiritiae radix, and Silybi mariani fructus. Ultrasound-assisted extraction was the
extraction method of choice due to shorter extraction time and superior extraction results over
maceration. Application of glycerol resulted in eco-friendly extracts with pronounced
antioxidant properties that inhibited selected skin-related enzymes. In addition to its many
advantages over conventional solvents, such as humectant properties and biocompatibility, that
eliminates the need to remove the solvent from the extract, glycerol was also active in the
protein coagulation assay thus displaying anti-inflammatory properties. The observed
biocompatibility with HaCaT cells indicated that the prepared glycerol extracts could be used
in dermatopharmaceutical preparations without prior removal of the solvent, thereby reducing
energy consumption and negative environmental impact. The observed biological effects of the
tested plant extracts indicate that they have a promising role in development of innovative
dermatopharmaceutical products intended for diminishing the consequences of skin aging.
Additional research is needed to determine the method of incorporation of the extracts into
dermopharmaceutical cosmetics, as well as their exact dosage.
Keywords
Comparison of methods for detecting antibodies in myasthenia gravis
No full textMiastenija gravis (MG) je autoimuna bolest karakterizirana slabošću i zamorom skeletnih mišića, a uzrokovana je prisutnošću autoantitijela na dijelove neuromišićne spojnice, najčešće na acetilkolinske receptore (AChR) i mišićno-specifičnu tirozin-kinazu (MuSK). Dijagnoza MG temelji se na kliničkoj slici i laboratorijskim testovima za otkrivanje autoantitijela. Cilj ovog rada bio je usporediti dvije metode detekcije autoantitijela – enzimsku imunosorbentnu analizu (ELISA) i metodu indirektne imunofluorescencije na transfeciranim stanicama (CBA IIF) te utvrditi njihovu podudarnost.
CBA IIF metodom je analizirano 77 ostatnih uzoraka seruma bolesnika preostalih nakon rutinskog laboratorijskog određivanja antitijela na AChR kvantitativnom ELISA metodom. Kvantitativnom ELISA metodom utvrđeno je da je 30 uzoraka negativno, 22 granično, a 25 pozitivno. Dobiveni
rezultati pokazali su razlike u osjetljivosti, specifičnosti i točnosti ELISA metode u ovisnosti jesu li granični rezultati dobiveni ELISA metodom promatrani kao pozitivni ili kao negativni. Također, rezultatima je pokazana i razlika u podudarnosti između ELISA metode i CBA IIF metode u ovisnosti promatranja graničnih rezultata kao pozitivnih ili kao negativnih. Osjetljivost ELISA metode značajno je viša kada se granični rezultati tumače kao pozitivni (100%), dok je u tom slučaju specifičnost značajno niža (50%). S druge strane, kada su granični rezultati ELISA metode tretirani kao negativni, osjetljivost se smanjuje na 88,24%, dok specifičnost raste na 83,33%. Točnost metode također je značajno viša kada se granični rezultati smatraju negativnima (84,42% naspram 61,04%). Cohen kapa koeficijent pokazuje da je podudarnost između ELISA metode i CBA IIF metode umjerena (κ = 0,61) kada se granični rezultati ELISA metode promatraju kao negativni, a kada se granični rezultati ELISA metode promatraju kao pozitivni, podudarnost među metodama je minimalna (κ = 0,31).
Zaključno, pri interpretaciji rezultata analize ELISA metodom, potrebno je uzeti u obzir način klasifikacije graničnih rezultata. Primjena CBA IIF metode kao potvrdne dijagnostičke metode doprinosi smanjenju broja lažno pozitivnih rezultata i povećanju specifičnosti dijagnostike MG-a.Myasthenia gravis (MG) is an autoimmune disease characterized by skeletal muscle weakness and fatigue, and is caused by the presence of autoantibodies to parts of the neuromuscular junction, most commonly to acetylcholine receptors (AChR) and muscle-specific tyrosine kinase (MuSK). The diagnosis of MG is based on clinical presentation and laboratory tests for detecting autoantibodies. The aim of this study was to compare two autoantibody detection methods – enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence on transfected cells (CBA IIF) – and to determine their concordance.
Using the CBA IIF method, 77 residual patient serum samples, remaining after routine laboratory quantification of AChR antibodies by ELISA, were analyzed. The quantitative ELISA method determined that 30 samples were negative, 22 borderline, and 25 positive. The obtained results revealed differences in sensitivity, specificity, and accuracy of the ELISA method depending on whether the borderline ELISA results were considered positive or negative. Additionally, the results demonstrated a difference in concordance between the ELISA and CBA IIF methods, depending on whether the borderline results were interpreted as positive or negative.
The sensitivity of the ELISA method was significantly higher when borderline results were interpreted as positive (100%), whereas, in this case, specificity was significantly lower (50%). On the other hand, when borderline ELISA results were considered negative, sensitivity decreased to 88.24%, while specificity
increased to 83.33%. The overall accuracy of the method was also significantly higher when borderline results were classified as negative (84.42% vs. 61.04%). The Cohen’s kappa coefficient indicated that the agreement between the ELISA and CBA IIF methods was moderate (κ = 0.61) when borderline ELISA results were considered negative, whereas the concordance between the methods was minimal (κ = 0.31) when borderline ELISA results were interpreted as positive.
In conclusion, when interpreting ELISA results, the classification of borderline results should be taken into account. The use of the CBA IIF method as a confirmatory diagnostic test contributes to reducing the number of false-positive results and increasing the specificity of MG diagnostics
Prirodne tvari u liječenju neuropatske boli
No full textCilj istraživanja
Neuropatska bol zahvaća 7-10 % opće populacije, a standardna farmakoterapija je učinkovita u manje od 50 % pacijenata i uzrokuje mnogo nuspojava koje ograničavaju njihovo korištenje. Cilj ovog specijalističkog rada je pretražiti kliničke znanstvene spoznaje o učinkovitosti i sigurnosti primjene odabranih prirodnih tvari u liječenju neuropatske boli kako bi se istražile nove učinkovite terapijske mogućnosti kod ove vrste boli.
Materijali i metode
Istraživanja u okviru ovoga specijalističkog rada teorijskog su karaktera i uključuju pregled dostupne literature o dosadašnjim suvremenim znanstvenim spoznajama o odabranim prirodnim tvarima u liječenju neuropatske boli. U pretraživanju radova relevantnih za temu ovoga rada korištene su elektronske bibliografske baze podataka PubMed (Medline), Science Direct, Scopus i Cochrane Library. Važan izvor informacija bile su i monografije i izvješća o ocjeni biljnih droga koje donosi Europska agencija za lijekove.
Rezultati
Potencijal u liječenju neuropatske boli, temeljem utvrđenih mehanizama djelovanja i pretkliničkih istraživanja, pokazale su sljedeće biljne tvari i vrste: kapsaicin, kanabinoidi, kurkumin, noćurak, šafran i gospina trava. Najviše kliničkih dokaza ima lokalna primjena kapsaicina u obliku flastera (8 %) kod liječenja postherpetične i dijabetičke neuropatije te neuropatije povezane s HIV-om. Kvaliteta dokaza je bila umjerena ili vrlo niska, uglavnom zbog malog broja istraživanja i nedovoljno ispitanika. Iako su provedena brojna istraživanja s pripravcima na bazi kanabinoida, dokazi o njihovoj učinkovitosti su nedostatni. Oromukozni sprej na bazi kanabinoida (2,7 mg THC/2,5 mg CBD) smanjuje bol i olakšava spavanje kod pacijenata s perifernom neuropatskom boli i centralnom neuropatijom kod multiple skleroze. Dvije studije su ukazale na povoljan učinak različitih formulacija kurkumina kod perifernih neuropatija. Također, kliničke studije koje bi potvrdile povoljan učinak noćurka, šafrana i gospine trave u liječenju neuropatske boli nedostaju (jedna do dvije relevantne studije).
Zaključak
Za navedene biljne tvari i vrste potrebno je provesti velike, dobro dizajnirane i kontrolirane studije kako bi se osigurali čvrsti dokazi o njihovoj mogućoj učinkovitosti i sigurnosti u liječenju neuropatske boli. Poticanje primjene prirodnih tvari u liječenju ove vrste boli može smanjiti upotrebu i nuspojave konvencionalnih lijekova te povećati kvalitetu života pacijenata.Objectives
The prevalence of neuropathic pain is estimated between 7 and 10 % in the general population and standard pharmacological treatments are effective in less than 50 % of patients and often are associated with undesirable side-effects that limit their use. The aim of this specialistic work is to research clinical scientific knowledge about efficacy and safety of selected natural products in the treatment of neuropathic pain in order to examine new therapeutic options for this type of pain.
Material and Methods
Research in this work is of theoretical character and includes an overview of the available literature on the up-to-date scientific knowledge about selected natural products in the treatment of neuropathic pain. Electronic bibliographic databases such as PubMed (Medline), Science Direct, Scopus and Cochrane Library were searched for relevant literature for this work. European Union herbal monographs and assessment reports were also an important source of information.
Results
Based on established mechanisms of action and preclinical research, these are natural products and plant species that have potential in treating neuropathic pain: capsaicin, cannabinoids, curcumin, evening primrose, saffron and St John's wort. Best evidence has local use of the capsaicin patch (8 %) in the treatment of postherpetic, diabetic neuropathy and neuropathy associated with HIV. Quality of the evidence was moderate or very low, mainly due to the small number of studies and insufficient number of participants. Despite a large number of studies with cannabinoids, evidence of efficacy is still lacking. Oromucosal spray with cannabinoids (2.7 mg THC/2.5 mg CBD) reduces the pain and sleep disorders in patients with peripheral neuropathic pain and central neuropathic pain in multiple sclerosis. Two studies have shown beneficial effects of different formulations of curcumin on peripheral neuropathies. Likewise, clinical studies that prove beneficial effects for evening primrose, saffron and St John's wort in treating neuropathic pain are lacking (one or two relevant studies).
Conclusion
Large, well-designed and controlled studies are needed for these herbal substances and species to provide strong evidence of their possible efficacy and safety in the treatment of neuropathic pain. Encouraging the use of natural products in the treatment of this type of pain can reduce the use and side effects of conventional drugs and increase the quality of life of patients
Isolation and composition of essential oil of southern wormwood (Artemisia abrotanum L.)
No full textU okviru ovog diplomskog rada istražen je sadržaj i sastav eteričnog ulja divljeg pelina (Artemsia
abrotanum L). Eterično ulje izolirano je metodom destilacije pomoću vodene pare. Određeno je da
cvatući vršni dijelovi sadrže 12,5 mL/kg eteričnog ulja. Analizom u spregnutom sustavu plinske
kromatografije i spektrometrije masa identificirano je 37 sastavnica, što predstavlja 86,73 %
eteričnog ulja. Među sastavnicama prevladavali su monoterpeni (64,06 %), dok je seskviterpenska
frakcija bila manje zastupljena (22,30 %). Prema stupnju oksidacije prisutnih monoterpena i
seskviterpena, ketonski su spojevi zauzeli prvo mjesto s udjelom od 49,09 %, a slijede ih
ugljikovodici (15,50 %), alkoholi (9,35 %), oksidi (6,54 %), esteri (3,66 %) i aldehidi (2,22 %).
Glavne sastavnice analiziranog eteričnog ulja bili su monoterpenski keton piperiton i seskviterpenski
keton davanon s udjelima 26,98 %, odnosno 13,22 %. Pojedinačnim udjelima istaknuli su se također
trans-pinokarveol (6,41 %), pinokarvon (5,96 %), α-pinen (5,63 %) i 1,8-cineol (4,45 %).In this diploma thesis, the content and composition of the essential oil of southern wormwood
(Artemsia abrotanum L). were studied. The essential oil was isolated by steam distillation. Its
content in the flowering tops was determined to be 12.5 mL/kg. Using a coupled system of gas
chromatography and mass spectrometry, 37 components were identified, accounting for 86.73% of
the essential oil. Among the components, monoterpenes predominated (64.06%), while the
sesquiterpene fraction was less abundant (22.30%). According to the degree of oxidation of the
monoterpenes and sesquiterpenes present, the ketone compounds ranked first with a percentage of
49.09%, followed by hydrocarbons (15.50%), alcohols (9.35%), oxides (6.54%), esters (3.66%) and
aldehydes (2.22%). The main components of the analyzed essential oil were monoterpene ketone
piperitone and sesquiterpene ketone davanone with proportions of 26.98% and 13.22%, respectively.
Trans-pinocarveol (6.41%), pinocarvone (5.96%), α-pinene (5.63%) and 1,8-cineole (4.45%) were
also highlighted with individual proportions