Project PN-II-PT-PCCA-2013-4-1572 (Contract. No. 161/2014)
Acronym: SALIFEROVEG
Studies on soil-plant-food-man correlations to obtain a food supplement with an increased content in iron of herbal origin
Project director: Prof. Robert Ancuceanu, RPh, PhD.
Financing source:
Romanian Ministry of National Education -UEFISCDI, the programme "Parteneriate in domenii prioritare - PN II ( Partnerships in priority sectors- PN II), Collaborative projects of applicative research
( http://uefiscdi.gov.ro/articole/2992/Proiecte-colaborative-de-cercetare-aplicativa.html - In Romanian language)
Project description
Iron deficiency anemia (IDA) is the most common form of anemia in the world and the most frequent form of anemia in pregnant women. It is estimated that IDA has a prevalence of 2% among adult men and 9-20% among adult women. The multiple negative effects of anemia on health and quality of life justify the interventions designed to prevent and control anemia and the most important such intervention is the use of iron-containing food supplements.
The core objective of this project is to develop an
iron-containing food supplement, using iron in its natural form, as
an extract from plants rich in this oligoelement and, for this
purpose, at exploring the correlations between soil, plants and the
developed food supplement, to ensure an optimal use in humans. There
are very few food supplements of botanical origin containing iron on
the Romanian market (most of those available use iron salts). There
is only a food supplement of Chinese origin containing iron (10
mg/tablet) and folic acid, the iron being derived from an extract of
the so-called "black fungi powder" (with no taxonomic
identification of the fungi species) and one manufactured locally (in
two slightly different formulas) but with very small amounts of iron
("Fier biologic", containing much less than 1.0 mg of Fe
per capsule).
For this purpose we would explore the correlation between soil iron and iron in plants, by analyzing iron contents in at least 10 plant species (for at least 2 parts of the plant species - root, stem, leaf, seeds) grown on various soils in Romania and using statistical methods of regression to verify the relationship between the two variables. Because - as shown in section 1.2. - iron bioavailability is most often affected by other components of the plants (especially phytates, polyphenols and flavonoids), we will also investigate the influence of the soils on the flavonoids and polyphenol contents (as phytates is most often found in significant amounts only in seeds and grains we will not investigate phytate).
Very little research has been concerned up to now to the
correlation between plant iron uptake and polyphenols or flavonoid
content in various plant parts. We will also explore the relationship
between the ontogenetic stages of plants and their iron uptake. The
studies published up to date have only looked into this relationship
in a small number of species and have produced inconsistent results.
These results will allow us to make a scientifically sound decision
on what parts of the plants to use, at what maturity stage to be
collected and grown on what soils, as starting material for an
extract rich in iron. We will also investigate the best iron
extraction method from herbal sources and the main variables
affecting the efficiency of the extraction process of iron in its
natural form from plants (the following variables will be
investigated: extraction method, solvent, temperature, herbal
product:solvent ratio, extraction time and particle size) and the
possibility of obtaining and characterizing a standardized extract
(to be used as a raw material for a finished product). Finally, we
will formulate and develop an iron-containing food supplement of
herbal origin at the prototype stage.
The project will create research synergies through the
collaboration of three partners: the University of Medicine and
Pharmacy "Carol Davila" Bucharest (with a multidisciplinary
team comprising experts in pharmaceutical botany, phytochemistry,
analytical chemistry, physical chemistry, pharmaceutical chemistry,
pharmacology, pharmaceutical technology)SC Hofigal Export Import, one
of the most important manufacturers of food supplements of herbal
origin on the Romanian market and SC Agroeco Bioterra SRL, a company
specialized in ecologically cultivating medicinal herbal species,
being independently certified in this sense.
The Project Team
1. Assoc. prof.Robert Ancuceanu, PhD - project director
2. Dr. Farm. Violeta Camen Popescu - team leader, S.C. Hofigal Import Export S.A.
3. Geta Stocheci - team leader, S.C. Agroecobioterra SRL
4. Prof. Mihaela Dinu, PhD (UMF „Carol Davila) - researcher
5. Prof. Valentina Uivaroşi, PhD (UMF „Carol Davila) - researcher
6. Assoc. prof.Alexandra Filareta Neagu, PhD (UMF „Carol Davila) - researcher
7. Prof. Cristina Dinu Pîrvu, PhD (UMF „Carol Davila) - researcher
8. Assoc. prof.Mircea Hîrjău, PhD (UMF „Carol Davila) - researcher
9. Lecturer Marilena Viorica Hovaneţ, PhD (UMF „Carol Davila) - researcher
10. Assist. prof. Adriana Iuliana Anghel, PhD (UMF „Carol Davila) - researcher
11. Assist. prof. Octavian Olaru, PhD (UMF „Carol Davila) - researcher
12. Assoc. prof.Corina Cristina Aramă, PhD (UMF „Carol Davila) - researcher
13. Lecturer Bruno Velescu, PhD (UMF „Carol Davila) - researcher
14. Assoc. prof.Mihai Niţulescu, PhD (UMF „Carol Davila) - researcher
15. Prof. Simona Negreş, PhD (UMF „Carol Davila) - researcher
16. Assoc. prof.Cornel Chiriţă, PhD (UMF „Carol Davila) - researcher
17. Assoc. prof.Corina Ana Ioniţă, PhD (UMF „Carol Davila) - researcher
18. Lecturer Elena Moroşan, PhD (UMF „Carol Davila) - researcher
19. Ec. Cecilia Niţă (UMF „Carol Davila) - technical staff
20. Ec. Alina Mihaela Ciudin - (UMF „Carol Davila) - technical staff
21. Alina Dune (S.C. Hofigal Import Export) - researcher
22. Florentina Steluta Dociu (S.C. Hofigal Import Export) - researcher
23. Maria Ciolea (S.C. Hofigal Import Export) - researcher
24. Emilia Tanasa (S.C. Hofigal Import Export) - technical staff
25. Larisa Petcu (S.C. Hofigal Import Export) - researcher
26. Mariana Fotescu (S.C. Hofigal Import Export) - researcher
27. Şerban Aurelia (S.C. Agroecobioterra SRL) - technical staff
28. Lenuţa Olaru (S.C. Agroecobioterra SRL) - technical staff
29. Costel Sahaidac (S.C. Agroecobioterra SRL) - technical staff
30. Ancuţa Negraru (S.C. Agroecobioterra SRL) - technical staff
31. Aurelia Zidaru (S.C. Agroecobioterra SRL) - technical staff
32. Elena Palangeanu (S.C. Agroecobioterra SRL) - technical staff
33. Ilie Coman (S.C. Agroecobioterra SRL) - technical staff
Expected results:
1. Understanding the correlations between soil characteristics and iron contents in the herbal species investigated.
2. Understanding the influence of the ontogenetic development stage on the iron contents in the herbal species investigated.
3. Understanding the correlations between the soil characteristics and the contents in minerals (other than iron) in the herbal species investigated
4.Understanding the influence of the ontogenetic development stage on the contents in minerals other than iron in the herbal species investigated
5. Understanding the correlations between the soil characteristics and the polyphenol contents in the herbal species investigated.
6. Understanding the influence of the ontogenetic development stage on the polyphenol contents in the species investigated.
7. Understanding the correlations between the soil characteristics and the flavonoid contents in the herbal species investigated.
8.Understanding the influence of the ontogenetic development stage on the flavonoid contents in the species studied.
9. Understanding the correlations between the characteristics of the soil and the contents in ascorbic acid in the species investigated.
10. Understanding the influence of the ontogenetic developmental stage on the ascorbic iron contents in the species investigated
11.Understanding the influence of the extraction method on the extraction yield (for iron, polyphenols, flavonoids and ascorbic acid)
12. Understanding the influence of the solvent on the extraction yield (for iron, polyphenols, flavonoids and ascorbic acid)
13. Understanding the influence of the temperature on the extraction yield (for iron, polyphenols, flavonoids and ascorbic acid)
14. Understanding the influence of the drug:solvent ratio on the extraction yield (for iron, polyphenols, flavonoids and ascorbic acid)
15. Understanding the influence of the extraction time on the extraction yield (for iron, polyphenols, flavonoids and ascorbic acid)
16. Understanding the influence of the particle size on the extraction yield (for iron, polyphenols, flavonoids and ascorbic acid)
17.Obtaining a standardizedherbal extract enriched in iron of herbal origin and its physico-chemical and microbiological characterization.
18. Assessment of the genotoxicity of the extract obtained on plant and animal cells..
19. Assessment of the bioavailability of iron using a validated in vitro method.
20. Obtaining a prototype of the finished product - a food supplement containing iron of herbal origin.
21.Dissemination: at least 3 papers index ISI Thomson and at least 3 communications to national/international conference; 1 national patent.
Stage I, 2014: Status and results obtained
The activities carried out within the Stage I/2014 of the project („Preliminary activities"), by the University of Medicine and Pharmacy "Carol Davila", as coordinator, and its two partners, S.C. Hofigal Export Import S.A. and S.C. Agroeco Bioterra S.R.L., had the following objectives:
1. Cultivation of the investigation plants (in open field or in greenhouse), collecting and/or processing the herbal material to be investigated, in order to be available for the other activities.
2. Checking/confirming the identity and purity of the herbal products used.
3. Developing and validating the assay method for iron and other oligoelements.
4. Developing and validating the assay method for polyphenols.
5. Developing and validating the assay method for flavonoids.
6. Developing and validating the assay method for ascorbic acid.
In this first stage preliminary activities were carried out, intended to facilitate the implementation of the complex experimental poject in the next stage.
1. Cultivation of the investigation plants (in open field or in greenhouse), collecting and/or processing the herbal material to be investigated, in order to be available for the other activities. Because the initiation of activities has taken place in the second part of the year, the herbal species necessary within the project could not be cultivated de novo, but dried herbal products cultivated by the partners in the period previous to the project kick-off were used, and preparations were made for the cultivation of the plants in the next stage.
2. Checking/confirming the identity and purity of the herbal products used. The criteria of macro- and microscopic identification of the herbal species which are the object of the project, the main histo-anatomic elements peculiar to each species and plant organ being verified and established. While macroscopical characters are easily accessible in the scientific literature, the microscopical ones are only partially available (especially for examinations on powders and on surface preparations).
3. Developing and validating the assay method for iron and other oligoelements. Iron and other oligoelements (zinc, chromium, lead, copper, manganese and cadmium) are assayed by atomic absorption spectrometry (AAS), according to the European Pharmacopoeia, the current edition. The validation was carried out on Liquiritiae radix (roots of Glycyrrhiza glabra L.). Linearity, repeatability and intermediate precision, accuracy and sensitivity of the method were confirmed. The specificity is going to be verified on each herbal product before the first application of the method to a certain product (analytical matrix).
4. Developing and validating the assay method for polyphenols. The spectrophotometric method with Folin-Ciocâlteu reagent (the gallic acid equivalence method) has been applied and validated. The validation was carried out on Liquiritiae folium (Glycyrrhiza glabra L. leaf), the leaf being considered a more complex analytical matrix than the root or stem. Linearity, repeatability and intermediate precision, accuracy and sensitivity of the method were confirmed. The specificity is going to be verified on each herbal product before the first application of the method to a certain product (analytical matrix).
5. Developing and validating the assay method for flavonoids. The method based on chelation with aluminium chloride in a slightly alkaline environment has been applied and validated, using quercetin (Merck) as a reference substance. The same parameters were also confirme for this method: linearity, repeatability and intermediate precision, accuracy and sensitivity.
6. Developing and validating the assay method for ascorbic acid . An HPLC method based on a stationary phase of the amino type (Microsorb 100-5 Amino, part. 5 mm) and a mobile phase consisting in a mixture of ortho-phosphoric acid 0,1% (A) and acetonitrile (B), was developed, the separation being performed in isocratic conditions (20% A, 80% B). For this method linearity and sensitivity were assessed. Because no herbal product derived from the 6 species investigated (at least 2 vegetative organs for each species) contained detectable amounts of ascorbic acid (the amounts were under the limit of detection) the precision and accuracy of the method were not investigated.
Stage II, 2015: Status and results obtained
The activities carried out within Stage 2/2015 of the project („The study of the influence of soil and ontogenetic development stage on the content in iron, polyphenols and ascorbic acid in plants – part 1”) by the University of Medicine and Pharmacy “Carol Davila” and its two consortium partners (S.C. Hofigal Export Import S.A. and S.C. Agroeco Bioterra S.R.L.) have been focused on the following objectives, with a view to investigating the influence of soil and ontogenetic development stage on iron and other minerals, polyphenols, flavones and ascorbic acid in plants:
1. Cultivation of the study plants, collection and/or processing of the herbal material to be used in the other activities (at least 6 species, at least 3 soils, at least 3 vegetation stages).
2. Checking/confirming the identity and purity of the herbal products used.
3. Identification and assay of iron from herbal products derived from different soils and collected at different development stages.
4. Identification and assay of other minerals from herbal products (first part).
5. Identification and assay of polyphenols in the herbal products.
6. Identification and assay of flavones in herbal products (first part).
The activities initiated within this stage will be finalized and integraged in the next stage of the project, allowing the development of a number of 10 experimental models (EM 1-10), describing the correlations between soil and ontogenetic development stage, respectively, on the one hand, and the content in iron, other minerals, polyphenols, flavones and ascorbic acid in the herbal species, in each organ.
1. Cultivation of the study plants, collection and/or processing of the herbal material to be used in the other activities (at least 6 species, at least 3 soils, at least 3 vegetation stages). Considering the objectives of this stage, a number of 7 herbal species have been cultivated in the conditions specified in the working protocol: cultivation on 3 different soils (a preluvosol with with clay loam, sandy texture and glomerular structure; a preluvosol with clay texture, rich in carbonates derived from carbonate rocks; a cernisol and chernozem soil of argic and cambic type, rich in humus) and collection at 3 different ontogenetic development stages (before flowering, at the bud stage and at anthesis).
2. Checking/confirming the identity and purity of the herbal products used. This has been carried out as in the first stage by the simple and classical methods of pharmacognostic analysis.Besides the previousspecies, additionally Plantago lanceolata L. has been included in the study, because due to eco-botanical reasons Acorus calamus L. was available for collection and analysis only in the first development stage.
3. Identification and assay of iron. This was carried out by atomic absorption spectrometry, according to the European Pharmacopoeia, the current edition, for the seven herbal species. In essence, a more limited influence of the soil was observed, as compared with the onto-genetic development stage (or the plant organ analyzed). Generally an increase in iron contents was seen during the first three onto-genetic development stages was seen, similar to the data reported in the literature for Phaseolus vulgaris, but differently from the data reported for Lactuca sativa.
4. Identification and assay of other minerals from herbal products (first part). Within this activity, other minerals (Ca, Mg, Na, K, Mn etc) were assayed in the herbal products derived from Tagetes erecta L. For the other species, the analyses are going to be performed in the next stage of the project.
5. For the polyphenol assay the spectrophotometric method using Folin-Ciocâlteu reagent (the method of gallic acid equivalence) was applied. The polyphenol content was assessed in all vegetative organs of the analyzed species, from the three soils, in the first three onto-genetic development stages (up to blossoming). The differences among development stages are generally more important than differences among soils and the variation between the development stages is not purely linear, but more complex, to be approached and understood distinctly for each species.
6. For the flavonoid assay, the method based on their chelation with aluminum chloride in a slightly alkaline environment was employed, using quercetin as a reference substance. The method was applied in this stage to a part of the herbal products derived from Urtica dioica, Tagetes erecta şi Glycyrrhiza glabra. The method was also attempted on flavonoids from Petroselinum crispum, but it could not be applied to any of the vegetative organs of the species, the absorbance of the solutions treated with this reagent being lower than that of the blanc (the yellow colour of the extractive solution being obviously paler for the test sample as compared with the control).
Stage III, 2016: Status and results obtained
The activities carried out within Stage 3/2016 of the project have been focused on the following objectives, with a view to investigating the influence of soil and ontogenetic development stage on iron and other minerals, polyphenols, flavones and ascorbic acid in plants, and the influence of the most important variables on the extraction yield:
1. Identification and assay of other minerals from herbal products (second part).
2. Identification and assay of flavones from herbal products (second part).
3. Identificarea and assay of ascorbic acid.
4. Chemical analyses of the soils off which the herbal material was collected.
5. The investigation of the factors influencing the extraction yield:
a. The extraction method
b. The solvent
c. The temperature
d. The solvent:herbal product ratio
e. The extraction duration
f. The particle size of the comminuted herbal product.
6. Cultivation of the plants to be studies, collecting and/or processing the herbal material for the other activities (at least 6 species, three soils, three stages of vegetation).
7. Checking/Confirming the identity and purity of the herbal products used.
The activities carried out in this stage, integrated with those from the precedent stage of the project implementation allowed the development of a number of 16 functional models (FM 1-16) describg the correlations between the soil and ontogenetic development stages on one side, and iron, other oligoelements, polyphenols, flavones and organic acids of the herbal species, in each vegetative organ.
1. Identification and assay of other minerals from herbal products (second part). Within this activity other minerals (Ca, Mg, Na, K, Mn etc) were assayed in the herbal products derived from Glycyrrhiza glabra L., Petroselinum crispum (Mill.) Fuss (sin. Petroselinum sativum Hoffm.), Plantago lanceolata L., Mentha x piperita L. and Acorus calamus L. There were significant and complex interactions between soil, development stage and iron or other mineral contents.
2. Flavonoid assay (second part) was continued for Urtica dioica, Tagetes erecta, Glycyrrhiza glabra and Petroselinum crispum (Mill.) Fuss (stages II and III), and fully carried out for Plantago lanceolata L., Mentha x piperita L., and Acorus calamus L. There were significant and complex interactions between soil, development stage and the flavonoid contents.
3. Identification and assay for ascorbic acid. As shown in the first stage, when validating the assay method for ascorbic acid, in all six herbal products the ascorbic acid is fully degraded, and consequently performing its assay is impossible. Therefore, taking into account that other organic acid (citric, malic etc) can influence the absorption of iron, we evaluated the contents inorganic acids in the aqueous extractive solutions from each of the herbal products analyzed, by measuring their pH. Among the species analyzed, the pH values tended to be lowest in Tagetes ercta L, Acorus calamus L. and Plantago lanceolata L. (most often under 6.00).
4. Chemical analyses of the soils off which the herbal material was collected were performed on a number of 12 samples - 4 for soil C, 5 for soil PC and 3 pentru soil PR – and were focused on the minerals also investigated in the hebral products studied within the project (Fe, Na, K, Ca, Mg, Cd, Cu, Cr, Mn, Zn). Notable differences were seen for a few elements (mainly Zn, Cu, Cd), especially in the PR soil, as compared with C and PC soils, the latter being more similar with respect to the elements analyzed.
5. The investigation of the factors influening the extraction yield (activities 3.5-3.10) has included the following variables, each with at least three distinct values/levels:
a. The extraction method (maceration, reflux, hot ultrasound extraction , and microwave extraction);
b. The solvent (six different solvents have been investigated: distilled water, methanol 50%, methanol 50% acidulated with citric acid 3%, methanol 50% acidulated with HCl 1N, water acidulated with citric acid 3%, and water acidulated with HCl 1N);
c. The temperature (three temperatures have been assessed for the reflux extraction: 65 °C, 80 °C, and 100 °C, as wellas room temperature - 20-25°C – for maceration, 40 hours);
d. The solvent: herbal product ratio (3 ratios were evaluated for reflux: 100:15, 100:5, and 100:2)
e. Duration of extraction (four duration of extraction by reflux have been evaluated: 10 minutes, 20 minutes, 30 minutes, 60 minutes).
f. The particle size (comminution degree) of the herbal product (3 size of the herbal powder were assessed: 20 μm, 100 μm, and 500 μm).
The results will be used in obtaining the final extract and first patented, then published.
6. Cultivation of the study plants (in field or in greenhouse), collection and/or processing of the herbal material for the carrying out of the other activities, also for the following year. A number of 7 herbal species have been cultivated in the conditions from the work protocol: cultivation on 3 different soils (a preluvosol with with clay loam, sandy texture and glomerular structure; a preluvosol with clay texture, rich in carbonates derived from carbonate rocks; a cernisol and chernozem soil of argic and cambic type, rich in humus) and collection at 3 different ontogenetic development stages (before flowering, at the bud stage and at anthesis).
7. Checking/confirming the identity and purity of the herbal products used. This has been carried out as in the first stage by the simple and classic methods of pharmacognositc analysis, as described in the previous report. In this stage mostly macroscopic examinations were used, and less microscopic; because we have acquired a Leica DMS 1000 digital microscope, we have analyzed each powder (without any previous clarification or processing) also with this microscope, so as to investigate the possibility of using this type of microscopy in the identification and purity assessment of the herbal products analyzed.
Stage IV - Status and results obtained
The activities carried out within this last stage of the project had the following objectives, oriented towards obtaining the standardized extract from the raw materials and a food supplement rich în iron of herbal origin (they are shown here în the order of activities assigned to this stage):
Obtaining the standardized extract (liquid, soft or dry extract; if liquid, possibly pulverized on a solid support as granules) and the assessment of the reproducibility of its method of obtaining
In vitro studies of geno- and cytotoxicity on the extract
Estimation of the iron bioavailability from the standardized extract using a validated in vitro method.
Preformulation and formulation studies.
The activities performed within this stage, integrated with those from the previous stages have allowed the development of a number of 5 experimental models:
a) Experimental model (EM1): obtaining an iron rich, standardized herbal extract
b) Experimental model (EM2): physico-chemical and microbiological characterization of the standardized extract
b) Experimental model (EM3): evaluation of the genotoxicity of the standardized extract (on plant cells)
c) Experimental model (EM4): evaluation of the citotoxicity of the standardized extract (on animal cells)
d) Experimental model (EM5): assessment of iron bioavailability using a validated in vitro method.
These have been the basis for building a Prototype of the finished product (Pi) – a food supplement containing iron of herbal origin.
1. Cultivation, collecting and/or processing the herbal material to be studied
Because based on the previous activities, a single species (Tagetes erecta L.) and a single herbal product (folium) derived from it were selected, in this stage only this herbal species was cultivated, on the three soil types that were previously described.
2. Checking/confirming the identity and purity of the herbal products used. For the same considerations, the checking/confirming the identity and purity were limited in this stage to this product only, using the simple and classical means of the pharmacognostic analysis, as showed previously.
3. 1. Obtaining the standardized extract (liquid, soft or dry extract; if liquid, possibly pulverized on a solid support as granules) and the assessment of the reproducibility of its method of obtaining. Based on the information provided by the functional models (FM1-FM9) built previously the leaves of Tagetes erecta L. were selected to obtain the standardized extract. The selection of this herbal product was based on its iron contents higher than in stem and root and on the renewable character of the leaves, which maybe collected without destroying the producing plant, unlike root. The solvent used to obtain the extract is water acidified with citric acid (3%), the solution obtained by refluxing the product with the solvent being then subjected to liophylization (after concentration). To assess the reproducibility of the method of obtaining the extract three experimental series of lyophylized extract were obtained from the T. erecta L. leaves, the yields being similar.
4. The physico-chemical control of the standardized extract is done by determining the loss on drying, iron, polyphenol and flavone assays, as well as by the specific extract IR spectrum. The determination of these parameters on three series of extract has indicated a relatively small variability from series to series and thus a good reproducibility of the obtaining method. The loss on drying of the lyophylized extract varied between 3.01 and 3.56%, the average iron contents was between 970.3 and 976.3 mg/kg, in polyphenols 2.80-2.92 (gallic acid equivalents), and in flavones of 0.367-0.372 mg/kg.
5. Microbiologic control of the standardized extract. Determination of the total number of aerobic microorganisms (TAMC) and of the total combined yeasts/ moulds count (TYMC) were performed on the basis of deep plate counts, as officialized in the European Pharmacopoeia, the edition currently in force. For the three series of extracts performed, the values obtained were within the compendial limits, being thus appropriate from the point of view of microbiological control.
6. In vitro studies of geno- and cyto-toxicity on the standardized extract. The assessment of toxicity on embrionary radicles of Triticum aestivum L. (and on the nucleus and chromosomes of the corresponding meristematic cells, respectively) and the assessment of the toxicity on nauplii of Artemia franciscana (Kellog, 1906), invertebrate crustaceans, were carried out. In both cases toxicity was seen at all concentration levels used (varying between 5% and 0.05%), an effect that is most likely related to the acidic pH created by the citric acid, present in abundance in the extract (because of the solvent used, and preserved in the extract for its biological effects of favouring iron absorption). This was confirmed by assessing the toxicitty of citric and ascorbic acids on embrionary radicles of T. aestivum L., at the same concentration levels as the extract; both natural substances were toxic at all levels tested. Literature data also reported cyto- and genotoxicity for the citric acid on human peripheral lymphocytes.
7. Estimation of iron bioavailability from the standardized extract based on an in vitro validated method. An in vitro model proposed first in 1981, which with slight modifications continue to be used nowadays, was used, being simple, rapid and with relatively low costs compared with the in vivo models. This used a stage of simulating gastric digestion (2 hours, digestion medium of 250 ml HCl 0.01 N containing 1.25 g pepsine, a receiving medium consisting of HCl 0.01 N) and one of simulating the intestinal digestion (2 hours, a digestion volume of 300 ml, consisting of 250 ml HCl 0.01 N, to which 40 ml 40 ml NaHCO3 0.5M and 10 ml solution of pancreatine and bile extract were added, and a receiving medium of NaHCO3 0.1M), and a dialysis membrane with a cut-off of 12,000 Da. The experiment simulating gastric absorption indicated that iron is dialyzed in a proportion of 49.90% through the semipermeable membrane, whereas the experiment simulating the intestinal digestion indicated an almost total diffusion of iron through the semipermeable membrane (94.80%).
8. Preformulation and formulation studies. To obtain the product capsules were chosen as the dosage form, taking into consideration the advantages that they have over other dosage forms. A formula was developed for capsules containing 500 mg T. erecta leaf L. lyophylized extract and maize starch, talc and magnezium stearate as excipients. Three pilot series were obtained based on the formula thus developed.
The dissemination of the research results was performed at this stage through a scientific paper and two communications at international conferences.
The protection of research results was carried out by submitting for approval to the Romanian State Office for Inventions and Trademarks for the herbal extract obtained.
Dissemination
1. Ancuceanu R, Aramă C, Neagu AF, Dinu M. Separarea şi dozarea acidului ascorbic prin HPLC: o analiză a literaturii ştiinţifice. Materialele Conferinţei Ştiinţifice Consacrate Jubileului de 50 de ani de la Fondarea Facultăţii de Farmacie şi 80 de ani de la Naşterea Patriarhului Farmaciei Moldave Vasile Procopişin (Revista Farmaceutică a Moldovei, 2014, nr. 3-4), p. 43
2. Ancuceanu VR, Arama C, Neagu AF, Dinu M, Hovaneţ MV, Olaru O, Popescu VC, Anghel AI. Ascorbic acid is virtually degraded in dried herbal products - an HPLC assessment of six plant species. Farmacia, 2015; 63 (5): 745-750
3. Ancuceanu R, Hovaneţ MV, Anghel AI, Olaru O, Popescu VC, Dinu M. Variation of polyphenol contents in vegetative organs of Tagetes erecta L. Priorities of Chemistry for a Sustainable Development, editia a XI - a Bucharest, 29th - 30th October 2015, International symposium - Book of Abstracts, BOOK OF ABSTRACTS, p. 13 (ISSN 2285 8334)
4. Ancuceanu, R.; Dinu, M.; Hovaneţ, M.V.; Anghel, A.I.; Popescu, C.V.; Negreş, S. A Survey of Plant Iron Content—A Semi-Systematic Review. Nutrients 2015; 7 (12): 10320-10351
5. Ancuceanu VR, Hovaneţ MV, Anghel AI, Dinu M, Dune A, Ciolea M, Olaru O, Popescu VC. Variation of polyphenols and iron concentration in Mentha x piperita L. by development stage and soil type. Paper accepted for publication by Farmacia.
6. R. Ancuceanu. Iron content of plants. Printech, Bucharest, 2016, 393 pages (book).
7. Ancuceanu VR, Dinu M, Hovaneţ MV, Olaru O, Popescu VC, Anghel AI. Dificultăţi şi probleme în dozarea flavonoidelor din produsele vegetale prin două metode bazate pe complexarea cu clorură de aluminiu. Congresul Naţional de Farmacie din România, ediţia a XVI-a. Farmacia - centru al interdisciplinarităţii ştiinţelor vieţii. Volum de Rezumate, Bucureşti, 2016.
8. 8. R. Ancuceanu, A. I. Anghel, M. V. Hovaneț, M. Dinu, O. T. Olaru, A. Dune, M. Ciolea, C.S. Stoicescu, C. Popescu. Variation of iron contents, polyphenols and flavonoids in Petroselinum crispum (Mill). Fuss (Apiaceae). In press, Farmacia (acceptance letter no. 33/29.09.2017).
9. A.I. Anghel, M.V. Hovaneț, M. Dinu, O.T. Olaru, Cristina S.Stoicescu, Carmen Popescu, Alina Dune, Robert Ancuceanu. Variation of mineral, polyphenol and flavonoid content by development stage in different parts of Urtica dioica l. Cultivated on three different soils. 3 rd International Conference on Natural Products Utilization: From Plants to Pharmacy Shelf ICNPU-2017, 18-21 October 2017 Bansko, BULGARIA (acceptance letter).
10. A.I. Anghel, M.V. Hovaneț, R. Ancuceanu, M. Dinu, C.S. Stoicescu, OT. Olaru, A. Dune, M. Ciolea, C. V. Popescu. Flavonoids, polyphenols and minerals from Acorus calamus L. (Acoraceae). PRIOCHEM 2017, Editia a XIII-a, 25-27 octombrie 2017, București (acceptance e-mail, 28.09.2017).