Methods for the determination of antioxidant activity of plant extracts in vitro

Plants are rich sources of secondary metabolites that exhibit diverse biological and pharmacological effects. Some plant ingredients, primarily phenolics, have significant in vitro antioxidant activity, which implies their contribution to the maintenance of redox balance in the body. These potential antioxidant agents are structurally very diverse, having different mechanisms of antioxidant activity. Since there is a growing necessity to detect, develop and understand effective antioxidant compounds, interest in the identification and the measurement of antioxidants in various plant isolates is persistently growing and many methods are being established. Most of the available in vitro tests are affordable and easy to perform, but due to the complex composition of plant extracts, different kinetics, mechanisms and specificity of the chemical reactions underlying these tests, there is no universal parameter for the assessment of antioxidant activity. In this paper, some of the currently most used in vitro methods for investigating and evaluating antioxidant activity of plant extracts are presented, emphasizing their advantages and weaknesses

Natural products: chemistry, bioactivity and application

The focus of research group from Department of Pharmacognosy at the Faculty of Pharmacy in Belgrade is on natural products, their chemistry and application. Main subjects of research are analysis of composition and bioactivities of various plant isolates (extracts, essential oils), isolation and structural analysis of active specialized plant metabolites, optimization of extraction procedures involving new techniques, and green chemistry principles. Since the Balkan Peninsula is one of the centers of plant biodiversity, our main objective is to explore the autochthonous taxa, especially endemic and rare ones, in terms of their distribution, conservation, morphology, chemistry, pharmacology and potential utilization. The main outcomes of these studies are the identification of new herbal raw materials and isolates, their pharmacognostic and pharmacological characterization, and the development of methods of quality assessment and evaluation of safe use of natural products, along with exploring other ways of their use. Important segment of research focuses on identifying natural principles and influences on secondary plant metabolism and studying the chemotaxonomic potential of certain plant metabolites. Basic research interest of this group is constantly upgraded trough scientific collaboration with colleagues from other research institution from Serbia, neighboring regions and abroad, resulting in many scientific and professional publications, and PhD theses.Oral presentation at "Training Program of Natural Products and Drug Discovery" jointly organized by Shanghai Institute of Materia Medica（SIMM), Chinese Academy of Sciences and Institute for Biological Research “Sinisa Stankovic”, National Institute of Republic of Serbia, University of Belgrade (IBISS) - 24-25 May, 2023, Belgrade

Biljni i tradicionalni lekovi u terapiji oboljenja srca i krvnih sudova

In the market there are various herbal medicinal products for supportive therapy of cardiovascular disorders. However, there are only few of such products, for which safety and efficacy have been adequately proven trough clinical studies. These are herbal medicinal products based on: standardized extracts of hawthorn leaf with flower, Crataegi folium cum flore (heart insufficiency: NYHA Stage II); standardized powdered garlic, Allii sativi bulbus (atherosclerosis); standardized extract of ginkgo leaf, Ginkgo bilobae folium (peripheral arterial occlusive disease: intermittent claudication), and standardized extract of horse chestnut seed, Hippocastani semen (chronic venous insufficiency). Other herbal medicinal products are mostly used based on their longstanding use and experience (traditional herbal medicinal products).Na tržištu se može naći veliki broj biljnih lekovitih proizvoda namenjenih za dopunsku terapiju različitih oboljenja kardiovaskularnog sistema. Međutim, mali je broj onih čije su efikasnost i bezbednost potvrđene odgovarajućim kliničkim studijama. U ovu grupu spadaju biljni lekovi na bazi standardizovanih ekstrakata lista sa cvetom gloga, Crataegi folium cum flore, koji se primenjuju kod blažih oblika srčane insuficijencije (NYHA klasa II), na bazi standardizovanog praška lukovice belog luka, Allii sativi bulbus, koji se koriste za prevenciju ateroskleroze, na bazi standardizovanog ekstrakta lista ginka, Ginkgo bilobae folium, namenjeni za simptomatsku terapiju okluzivne bolesti perifernih arterija (intermitentna klaudikacija) i na bazi standardizovanog ekstrakta semena divljeg kestena, Hippocastani semen, namenjeni za simptomatsku terapiji hronične venske insuficijencije. Primena ostalih biljnih lekovitih proizvoda uglavnom je zasnovana na dugoj tradiciji i iskustvu (spadaju u kategoriju tradicionalnih biljnih lekova)

Primena biljnih lekovitih proizvoda kod hronične venske bolesti

In treatment of chronic venous disease (CVD) venoactive drugs (VADs) are used. They represent a heterogeneous group of drugs of synthetic (ie. calcium dobesilate, naftazone, and benzarone) or natural origin. Venoactive drugs of herbal origin are usually prepared from isolated plant constituents (i.e. flavonoids) or their semisynthetic derivatives (i.e. hydroxyethylrutosides, diosmin), but also from various herbal drugs and their preparations (herbal medicinal products, HMPs). Herbal medicinal products used in these indications are usually prepared from: horsechestnut seed and bark (Hippocastani semen/cortex), butcher’s broom rhizome (Rusci rhizoma), melilot herb (Meliloti herba), grapevine leaf (Vitis viniferae folium), billberry fruit (Myrtilli fructus), French maritime pine bark (Pini pinasteris cortex), centella herb (Centellae asiaticae herba), and ginkgo leaf (Ginkgonis folium). Active compounds of these herbal drugs belong to groups of saponins, coumarines, flavonoids, anthocyanins, proanthocyanidins or terpenes. According to community herbal monographs prepared by Committee on Herbal Medicinal Products at the European Medicines Agency (EMA/HMPC), HMPs with well- established use are those prepared from dry standardized horsechestnut seed extract (with 16­28% of aescin) and dry standardized grapevine leaf water extract (DER 4-6:1), for oral use. Herbal medicinal products classified under traditional medicinal use provisions are products prepared from horsechestnut seed and bark, grapevine leaf, butcher’s broom rhizome, and melilot herb (for oral and/or cutaneous use).Za terapiju primarne hronične venske bolesti (HVB) koriste se venoaktivni lekovi. Predstavljaju heterogenu grupu lekova sintetskog (npr. Ca-dobesilat, naftazon, benzaron) ili prirodnog porekla. Venoaktivni lekovi biljnog porekla se uglavnom izrađuju od izolovanih biljnih sastojaka (najčešće flavonoida) ili njihovih polusintetskih derivata (npr. hidroksietilrutozidi, diosmin), ali i od različitih biljnih droga i njihovih preparata (biljni lekoviti proizvodi, BLP). Najznačajnije biljne droge od kojih se izrađuju BLP za primenu u ovom indikacionom području su: seme i kora divljeg kestena (Hippocastani semen/cortex), rizom kostrike (Rusci rhizoma), herba ždraljevine (Meliloti herba), list vinove loze (Vitis viniferae folium), plod borovnice (Myrtilli fructus), kora francuskog primorskog bora (Pini pinasteris cortex), herba centele (Centellae asiaticae herba), i list ginka (Ginkgonis folium). Aktivni sastojci ovih droga spadaju u grupu saponozida, kumarina, flavonoida, antocijana, proantocijanidina ili terpena. Prema monografijama biljnih droga koje je do sada pripremio Komitet za biljne lekovite proizvode Evropske agencije za lekove (EMA/HMPC), status biljnih lekova imaju oni BLP koji se izrađuju od suvog standardizovanog ekstrakta semena divljeg kestena (sadrži 16-28% escina) i suvog standardizovanog vodenog ekstrakta lista vinove loze (DER 4-6:1), i namenjeni su za oralnu primenu. Status tradicionalnih biljnih lekova imaju odgovarajući lekoviti proizvodi na bazi semena i kore divljeg kestena, lista vinove loze, rizoma kostrike i herbe ždraljevine (za oralnu i/ili dermalnu primenu)

Chemical composition and anti‐DPPH activity of essential oil obtained from commercial sample of Cretan dittany herb, Origani dictamni herba

Kritski origano, Origanum dictamnus L. (Lamiaceae), je endemična biljka grčkog ostrva Krit. Komitet za biljne lekovite proizvode Evropske agencije za lekove (EMA/HMPC) izdao je EU monografiju za tradicionalnu primenu herbe kritskog origana, Origani dictamni herba. Prema ovoj monografiji usitnjena herba se koristi oralno u obliku čaja (infuza) za ublažavanje kašlja povezanog sa prehladom i za simptomatsko ublažavanje blagog gastrointestinalnog diskomfora, kao i dermalno u obliku infuza ili dekokta za tretman manjih inflamacija kože i modrica. U Evropskoj farmakopeji (Ph. Eur.) za sada ne postoje monografije za ovu biljnu drogu i njeno etarsko ulje. Cilj ovog rada bio je ispitivanje hemijskog sastava etarskog ulja izolovanog iz komercijalnog uzorka herbe kritskog origana poreklom iz Grčke, kao i procena njegove antiradikalske aktivnosti. Etarsko ulje je izolovano iz herbe destilacijom vodenom parom u aparaturi po Clevenger-u, prema postupku Ph. Eur. Prinos etarskog ulja iznosio je 1,51% V/m, tj. 1,46% m/m. Hemijski sastav etarskog ulja analiziran je gasnom hromatografijom sa FID i MS detekcijom (GC-FID i GC-MS). Identifikovane su 42 komponente, koje su činile 94,6% sastava etarskog ulja. Procentualno najveći udeo u etarskom ulju činili su oksidovani monoterpeni (58,8%; 9 jedinjenja), a sledili su monoterpenski ugljovodonici (27,4%; 12 jedinjenja), seskviterpenski ugljovodonici (5,6%; 12 jedinjenja) i oksidovani seskviterpeni (2,3%; 6 jedinjenja). Glavna komponenta etarskog ulja bio je monoterpenski fenol karvakrol (51,4%), dok su po količini sledili monoterpenski ugljovodonici p-cimen (14,3%) i γ-terpinen (9,3%). Dobijeni rezultati bili su u skladu sa literaturnim podacima, pri čemu je u slučaju uzorka analiziranog u ovom radu, prinos etarskog ulja bio veći od prethodno publikovanih vrednosti (1, 2). Antiradikalska aktivnost izolovanog etarskog ulja ispitana je spektrofotometrijskim DPPH testom. Stepen antiradikalske aktivnosti je rastao linearno sa povećanjem koncentracije etarskog ulja, pri čemu je koncentracija u kojoj je etarsko ulje inhibiralo 50% DPPH radikala (SC50) iznosila 141,77 μg/mL. Značajan anti-DPPH potencijal etarskog ulja herbe kritskog origana može se barem delom objasniti velikim udelom karvakrola, čiji je antiradikalski potencijal ranije pokazan (3). Rezultati sprovedenih hemijskih ispitivanja mogu biti od značaja za buduće definisanje parametara farmaceutskog kvaliteta droge Origani dictamni herba i njenog etarskog ulja.Cretan dittany, Origanum dictamnus L. (Lamiaceae), is an endemic species from the Greek island of Crete. The Committee on Herbal Medicinal Products of the European Medicines Agency (EMA/HMPC) adopted EU monograph on traditional use of Cretan dittany herb, Origani dictamni herba. According to this monograph, comminuted herb is used orally as herbal tea (infusion) for the relief of cough associated with cold and for mild gastrointestinal disorders, as well as cutaneously as infusion or decoction for the treatment of minor skin inflammations and bruises. European Pharmacopoeia (Ph. Eur.) monographs for this herbal drug and its essential oil do not exist for now. The aim of this work was to investigate chemical composition of the essential oil obtained from a commercial sample of Cretan dittany herb and to evaluate its antiradical activity. The essential oil was isolated from the herb by hydrodistillation in a Clevenger‐type apparatus, according to procedure given in Ph. Eur. The yield was 1.51% V/w, i.e. 1.46% w/w. Chemical composition of the essential oil was analyzed by gas chromatography with FID and MS detection (GC-FID and GC-MS). A total of 42 components were identified, comprising 94.6% of the composition of the essential oil. The dominant were oxygenated monoterpenes (58.8%; 9 compounds), followed by monoterpene hydrocarbons (27.4%; 12 compounds), sesquiterpene hydrocarbons (5.6%; 12 compounds) and oxygenated sesquiterpenes (2.3%; 6 compounds). The main component of the essential oil was monoterpene phenol carvacrol (51.4%), followed by monoterpene hydrocarbons p-cymene (14.3%) and γ-terpinene (9.3%). Obtained results were in accordance with the literature data; it should be noted that in the case of the sample analyzed in this work, the essential oil yield was higher than previously published values (1, 2). The antiradical activity of the essential oil was investigated by spectrophotometric DPPH test. The percentage of antiradical activity increased linearly with the increase of essential oil concentration. The concentration at which the essential oil inhibited 50% DPPH radicals (SC50) was 141.77 μg/mL. Significant anti-DPPH potential of the essential oil of Cretan dittany herb can be at least partly explained by the presence of high quantity of carvacrol, which antiradical potential was demonstrated previously (3). The results of performed chemical investigations may be important for defining pharmaceutical quality parameters of the herbal drug Origani dictamni herba and its essential oil in the future.Drugi naučni simpozijum Saveza farmaceutskih udruženja Srbije sa međunarodnim učešćem, 28. 10. 2021. Beogra

Biljni lekoviti proizvodi: Uslovi za bezbednu primenu

As the use of herbal medicinal products is constantly growing, their safety became an important public health issue. In spite of common belief that herbal medicinal products are safe and harmless because they are natural, it is not always so and their usage is not devoid of certain risks. The most common reasons for herbal adverse reactions (ADRs) are: improper use (overdose or prolonged use), interactions with conventional drugs and poor quality of herbal products. In this work were discussed the most important issues on their safe usage. Classification of herbal medicinal products (herbal medicine, traditional herbal medicine) and basic informations on their registration and marketing authorization according to current European and Serbian legislation were also given. Manufacturing of quality herbal medicinal products is highlighted as one of the most important requirements for their safety. Having in mind that most of these products are sold without prescription (OTC) and that they are mostly used in self-medication, the role of health professionals in consulting patients on choosing of herbal medicinal product and its usage was emphasized. The importance of patient's collaboration with health professionals on recognizing and monitoring herbal ADRs and avoiding possible interactions was also pointed out. Examples of the most frequent herbal medicinal products ADRs occurring in course of their recommended and improper use, on their interactions with drugs as well as on their poor quality were given. The importance of involving herbal medicinal products in international pharmacovigilance systems was also explained

Koncept održive ishrane

Sustainable nutrition can be characterized as a diet with low environmental impact that is in accordance with good health, which emphasizes at the same time the nutritional adequacy of such a diet to achieve the general health of the population and its ecological acceptability. The impact of food production and nutrition on the environment occurs in all phases, from food production to household, but also during the deposition of waste generated in the production process or in households, and all of these food-related processes are responsible for emissions of 15-30% of the total GHGs emission. The results of numerous studies have indicated that transition towards more sustainable food consumption requires the replacement of the most animal proteins by plant-derived proteins, because plant-based food have a lower environmental impact as well as a positive effect on health. In recent years, great efforts have been made to implement the concept of sustainability into national food-based dietary guidelines, which primarily requires the awareness and support of national regulatory authorities. Finally, it should be noted that the Republic of Serbia is one of the few countries in Europe that hasn’t yet developed national food-based dietary guidelines, moreover it is the leading country in Europe when it comes to the incidence of the chronic non-communicable diseases. In line with the latest global requirements, effort is needed in the future to develop a national dietary guidelines that will have an integrated concept of sustainability.Održiva ishrana se može pre svega okarakterisati kao ishrana sa malim uticajem na životnu sredinu koja je u skladu sa dobrim zdravljem, čime se akcenat stavlja istovremeno i na nutritivnu adekvatnost takve ishrane za postizanje opšteg zdravlja populacije ali i na njenu ekološku prihvatljivost. Uticaj proizvodnje hrane, ali i ishrane, na životnu sredinu javlja u svim segmentima, počev od procesa proizvodnje do domaćinstva, ali i tokom deponovanja otpada koji takođe nastaje duže celog lanca. Svi ovi procesi u vezi sa hranom su odgovorni za emisiju čak 15–30% od ukupne emisije gasova sa efektom staklene bašte. Rezultati brojnih istraživanja ukazuju da je ključ koncepta održive ishrane zamena većeg dela životinjskih proteina proteinima iz biljnih izvora upravo zato što namirnice biljnog porekla imaju manji uticaj na životnu sredinu, a ujedno imaju i protektivni efekat na zdravlje. Poslednjih godina se ulažu veliki napori da se koncept održivosti implementira i u nacionalne dijetarne vodiče za šta je pre svega potrebna svest i podrška nacionalnih regulatornih tela. Na kraju treba istaći da je Republika Srbija jedna od retkih zemalja u Evropi koja još uvek nema dijetarni vodič za pravilnu ishranu, a pri tome je vodeća zemlja u Evropi po broju obolelih od hroničnih nezaraznih bolesti. U skladu sa najnovijim globalnim zahtevima, potrebno je u budućnosti raditi na razvijanju nacionalnog vodiča koji će imati integrisan i koncept održivost

Structural elucidation of flavonoids isolated from aerial parts of Stachys annua (L.) L. (Lamiaceae)

Stachys annua (L.) L. (annual yellow-woundwort) is an arrable weed and melliferous archaeophyte, with the native range in Europe to Siberia and Iran [1]. The aim of this work is to implement simple chromatographic technique for isolation of flavonoids from crude methanol extract of S. annua aerial flowering parts (herb). Plant material collected in NW Serbia (Mt Sokolske planine) was air-dried, powdered, and then extracted by bimaceration with chloroform and methanol. Dry methanol extract of S. annua herb was applied on preparative TLC plates of silica gel. After developing of chromatogram in methylene chloride:methanol:water (40:10:1, V/V/V), and spraying with NP/PEG reagent, in ultraviolet light at 365 nm three clearly separated fluorescence-quenching zones were observed. Zones were eluted with mixture of methylene chloride and methanol (1:1, V/V), and using LC-MS analysis one dominant compound was detected in each of these fractions. According to their UV, MS and 1H-NMR spectral data, as well as using flavonoids previously isolated from S. alpina subsp. dinarica [2], these compounds (1-3) were identified as 8-hydroxyflavone glycosides, namely: isoscutellarein-7-O-[6'''-acetyl-β-D-allopyranosyl-(1→2)]-β-D-glucopyranoside (compound 1), 3'-hydroxy-4'-O-methylisoscutellareine-7-O-[6'''-acetyl-β-D-allopyranosyl-(1→2)]-β-D-glucopyranoside (compound 2) and 4'-O-methylisoscutellareine-7-O-[6'''-acetyl-β-D-allopyranosyl-(1→2)]-β-D-glucopyranoside (compound 3). It could be concluded that the developed preparative TLC method is suitable for rapid isolation of the mentioned flavonoid biosides from the crude methanol extract of S. annua without prior fractionation by other chromatographic techniques.5th International Conference on Natural Products Utilization: from Plants to Pharmacy Shelf - ICNPU-2023, Sts. Constantine & Helena, Bulgaria, 30th May to 2nd June 202

Amino acids composition in selected nuts

In recent years, we are witnessed that a nuts very popular food and is recommended as part of a diet because it contains numerous bioactive compounds. Nuts contain a high quantity of protein, i.e. amino acids that are known to be necessary for important processes such as tissue growth, energy production, immune function and nutrient absorption. In order to check the amount of individual amino acids found in the favorite samples of nuts of our citizens, we determined total protein and amino acid profile (17 amino acids) in nine samples of nuts (peanut, almond, hazelnut, walnut, Brazil nut, cashew, pecan, pistachio, and pine nuts). Total amino acids composition was determined by ion chromatograph with electrochemical detector, manufactured by Thermo, model ICS-5000, with silver reference electrode (Ag / AgCl) and gold (Au) working electrode and chromatographic column AminoPac PA10 and pre-column AminoPac PA10 guard. Analyzed nuts samples contained 11.7-29.2 g/100 g total proteins. Pecan contained the least protein, while peanut was the richest in total protein. Among the amino acids, glutamic acid, aspartic acid and arginine contents were generally high, whereas methionine, cysteine, histidine and tyrosine contents were low. Peanut was the most important source for essential amino acids (lysine 1.08 g/100 g; histidine 0.71 g/100 g; threonine 0.87 g/100 g; leucine 1.97 g/100 g; isoleucine 1.25 g/100 g; valine 1.07 g/100 g; phenylalanine 1,38 g/100 g; and methionine 0.4 g/100 g). Cashew was the biggest source of essential amino acids, 37% (essential/total amino acids), while the smallest source was almond, 27%. Almond was the biggest source of non-essential amino acids, 73%. The obtained results indicate that the nuts are incomplete in the content of amino acids. Knowledge of the amino acid composition of nuts is essential for combination with other food groups when formulating a balanced diet.UNIFood2021 Conference, 24th-25th September 2021 University of Belgrade, 2nd International UNIfood Conferenc

Quality characteristics of oils from selected edible seeds

Many types of vegetable oils produce around the world. They are useful in nutrition, prevention and treatment of diseases. Some oils traditionally have been well known for a long time and have been shown many potential health benefits, such as, cardioprotective effect, effect on digestion, risk of some cancer and it is also used in the treatment of skin infections. This work was conducted to study some quality criteria of oil extracted from seven different raw seeds purchased in the Serbian market, namely: sunflowers, sesame seeds, black sesame seeds, tickles seeds, chia seeds, flax seeds and hemp seeds. Oils are essentially regarded in terms of quality based on their free fatty acid, iodine value and peroxides value. Oils were extracted from the seeds by cold extraction with petroleum ether. Quality characteristics, such as refractive index, acid value, peroxide value, iodine value and saponification value and unsaponifiable matter were determined. For the determination of these parameters, official methods (ISO 6320; ISO660; ISO 3960; ISO 3961; ISO 3657 and ISO 18609, respectively) were used. The contents of investigated parameters in seed samples were determined in the range of 1.467 to 1.485 nD for refractive index; 1.62 to 4.95 mg KOH/g for acid value; 5.21 to 7.45 for peroxide value; 118 to 197 for iodine value; 180 to 193 for saponification value and 3.14 to 7.26 g/kg for the unsaponifiable matter. Data obtained in this investigation indicate that all quality parameters of the seed samples, except peroxide value , comply with the national and Codex standards.10th International Congress of Food Technologists, Biotechnologists and Nutritionists, 30.11-2.12.2022. Zagreb, Croati