UNIVERSITY OF MEDICINE AND PHARMACY CRAIOVA

Ph.D. THESIS PHARMACOGNOSTICAL AND PHARMACOLOGICAL RESEARCHES ON SOME VEGETAL SPECIES WITH HYPOGLYCEMIC VIRTUES
ABSTRACT

Scientific Supervisor Professor of Pharmacology Florica Popescu, Ph. D Ph.D. Candidate Anca Berbecaru-Iovan

CRAIOVA 2009

SUMMARY

1. Introduction
STATE OF KNOWLEDGE

2. Pharmacognostical and pharmacological data on some vegetal species with hypoglycemic activity 3. General information about diabetes mellitus
PERSONAL CONTRIBUTION

4. Research on obtaining and physical and chemical characterization of tinctures of hypoglycemic plants 4. 1. Introduction 4. 2. The determination of the identity, purity and quality of plants material 4. 3. Preparation of tinctures by simple percolation method 4. 4. Organoleptic rating 4. 5. Determination of relative density 4. 6. Determination of refractive index 4. 7. Quality conditions 4. 8. Quantitative determination of phenyl-propane compounds 4. 8. 1. Materials and methods 4. 8. 2. Results and discussions 4. 9. Flavonoids and polyphenolic acids analysis by TLC 4. 9. 1. Materials and methods 4. 9. 2. Results and discussions 4. 10. VIZ footprint spectrophotometric 4. 10. 1. Materials and methods 4. 10. 2. Results and discussions 4. 11. IR spectral footprint 4. 11. 1. Materials and methods 4. 11. 2. Results and discussions 4. 12. Conclusions 5. Preliminary studies on hypoglycemic effect of plant species 5. 1. Preliminary studies on hypoglycemic effect of plant species on experimentally induced hyperglycemia by adrenaline in rats 5. 1. 1. Introduction 5. 1. 2. Materials and methods 5. 1. 3. Results and discussions 5. 1. 4. Conclusions 5. 2. Preliminary studies on the evaluation of Syringae vulgaris flos f. lba and Syringae vulgaris flos f. violcea tinctures on blood glucose levels when performed oral glucose tolerance test in normal rats

5. 2. 1. Introduction 5. 2. 2. Materials and methods 5. 2. 3. Results and discussions

5. 2. 4. Conclusions
5. 3. Acute and subacute toxicity study of Syringae vulgaris flos f. lba and Syringae vulgaris flos f. violcea tinctures 5. 3. 1. Introduction 5. 3. 2. Materials and methods 5. 3. 3. Results and discussions

5. 3. 4. Conclusions
6. Experimental model to induce diabetes by administration of streptozotocin in rat 6. 1. Introduction 6. 2. Materials and methods 6. 3. Results and discussions

6. 4. Conclusions
7. Evaluation of hypoglycemic effect of some vegetal species on streptozotocin-induced diabetes in rats 7. 1. Introduction 7. 2. Materials and methods 7. 3. Results and discussions

7. 4. Conclusions
8. Research on the hypoglycemic effect, cholesterol-lowering and antioxidant effect of plant species in rats with experimental streptozotocin-induced diabetes 8. 1. Introduction 8. 2. Materials and methods 8. 3. Results and discussions

8. 4. Conclusions
9. Final conclusions 10. Bibliography Key words: diabetes mellitus, phytotherapy, vegetal species, hypoglycemic effect, cholesterol lowering effect, antioxidant effect

1. INTRODUCTION The worldwide prevalence of diabetes mellitus (DM) has risen dramatically over the past two decades; based on current trends, more than 360 million individuals will have diabetes by year 2030. In addition to oral agents and insulin therapy, phytotherapy is an alternative that offers a wide range of natural resources with hypoglycemic effects some plants provide materials recommended for people with diabetes. Herbal remedies are beneficial to patients with type 2 diabetes and, in addition to the diet prescribed by a doctor, they can help glucose homeostasis, but they can not replace insulin and oral medication. In recent years, researchers turned attention specifically to oxidative stress and the key role it plays, as a common element in the pathogenesis of diabetes complications. Hyperglycemia generates reactive oxygen species which, in turn, causes membrane lipid peroxidation and degradation. Many of the complications of diabetes, including vascular atherosclerosis, major cause of mortality in DM, are closely related to oxidative stress and, thus, antioxidants play an important role in the treatment of diabetes. Herbal remedies contain large amounts of antioxidants such as flavonoids, polyphenolic acids, carotenoids, vitamins C and E; experimental research has shown that the antioxidant activity may be an important property of medicinal plants used for their hypoglycemic effect in the treatment of DM. STATE OF KNOWLEDGE 2. Pharmacognostical and pharmacological data on some vegetal species with hypoglycemic activity Plants with hypoglycemic action from the Romanias spontaneous flora are mostly from families: Asteraceae, Cruciferae, Ericaceae, Fabaceae, Gentianaceae, Lamiaceae, Liliaceae, Moraceae, Polygonaceae, Rosaceae. In terms of distribution of active ingredients, most hypoglycemic medicinal plants include: flavonoids, polyphenolcarboxylic acids, sterols and triterpenoid, tannins, volatile oils, saponozide, bitter principles, coumarines, carotenoids, mucilages, micronutrients, alkaloids, aminoacids, proteins, the therapeutic activity being the result of interaction of several classes of compounds, respectively of an phytocomplex.

Pharmaceutical preparations based on natural products recommended in DM may contain either a single plant product (powder or extract) or combination of products with hypoglycemic action or other properties beneficial to the diabetic patient. 3. General information about diabetes mellitus Diabetes mellitus refers to a group of common metabolic disorders that is characterized by hyperglycemia. Depending on the etiology of diabetes, factors leading to hyperglycemia include decreased insulin secretion, decreased glucose use and increased production of glucose. The metabolic dysregulation associated with DM causes secondary pathophysiologic changes in multiple organ system that imposes a tremendous burden on the individual with diabetes and on health system. DM is the leading cause of end-stage renal disease, nontraumatic lower extremity amputation and adult blind. Antidiabetic medication seeks to maintain a normal glucose level. Antidiabetic drug classification by origin, chemical structure and mechanism of action includes insulin and insulin analogues and oral antidiabetic agents used in type 2 diabetes. The oral antihyperglycemic drugs may enhance pancreatic insulin secretion (secretagogues), sensitize peripheral tissues to insulin (sensitizers) and impair GI absorption of glucose. PERSONAL CONTRIBUTION 4. Research on obtaining and physical and chemical characterization of tinctures of hypoglycemic plants The main objectives of this chapter were the obtaining and the physical and chemical characterization of tinctures of hypoglycemic plant products studied (Cynosbati fructus, Galegae herba, Myrtilli folium, Myrtilli fructus, Phaseoli sine seminibus fructus (pericarpium), Syringae vulgaris flos f. lba, Syringae vulgaris flos f. violcea, Trigonellae foenumgraeci semen, Violae tricoloris herba); qualitative and quantitative analysis for phenyl-propan compounds from the analised tinctures by thin layer chromatography (TLC) and spectrophotometric VIZ; flavonoids and polyphenolic acids analysis to separate and identify them, using appropriate standards, by TLC. Analysis of chromatograms obtained confirmed flavonoids and polyphenolic acids presence in all samples analyzed except the tinctures

from Trigonellae foenum-graeci semen and Galegae herba, where was no evidence of polyphenolic acids. For the first time were characterized as such, without further processing, both in terms of absorption peaks and pads in VIZ and specific vibration in IR the tinctures obtained by simple percolation of the nine hypoglycemiant plant products, the results contributing to the schemes which can identify various tinctures and determine their degree of alteration. 5. Preliminary studies on hypoglycemic effect of plant species 5. 1. Preliminary studies on hypoglycemic effect of plant species on experimentally induced hyperglycemia by adrenaline in rats The aim of this study was to investigate the hypoglycemic effect of the powder of mulberry leaves (Mori folium) after oral administration in rats with experimentally-induced adrenalin hyperglycemia, compared with less studied species regarding their hypoglycemic effect, pansy (Violae tricoloris herba) and acacia leaves (Robiniae pseudoacaciae folium). The results showed that the Mori folium powder easily decrease the experimental hyperglycemia induced by adrenalin in rats and promote a return to baseline of the blood glucose level, proving to be a remedy with mild hypoglycemic activity. Robiniae pseudoacaciae folium and Violae tricoloris herba powders marginally reduced hyperglycemia in rats compared with control, but favoring a return of glucose values close to the reference. 5. 2. Preliminary studies on the evaluation of Syringae vulgaris flos f. lba and Syringae vulgaris flos f. violcea tinctures on blood glucose levels when performed oral glucose tolerance test in normal rats This research aimed to investigate the potential antihyperglycemic effect on oral glucose tolerance test (OGTT) of tinctures of Syringae vulgaris flos f. lba and Syringae vulgaris flos f. violcea when given orally to normal rats one hour prior to the OGTT. Improving response to exogenous glucose load, performed after the treatment with tinctures, is demonstrated by a decrease in blood glucose level at both 30 and 60 minutes, compared with corresponding values from untreated group (control). This pharmacodynamic effect was not shown so far from the studied plant products.

5. 3. Acute and subacute toxicity study of Syringae vulgaris flos f. lba and Syringae vulgaris flos f. violcea tinctures This study aimed to asses the toxic potential after oral administration of tincture of Syringae vulgaris flos f. alba and Syringae vulgaris flos f. violcea by conducting in vivo experiments in normal rats. In determining the toxicity degree of the studied plant products, were used much higher doses than the therapeutic doses, watching the emergence of toxic and lethal effects at these doses. Research conducted on normal rats showed that no animal has died during the study. Treated animal behaviour was normal and there was no toxic and lethal effects reported even at doses of 10 or 20 times higher than the effective dose of tincture of Syringae vulgaris flos f. lba, respectively Syringae vulgaris flos f. violcea. 6. Experimental model to induce diabetes by administration of streptozotocin in rat In addition to presenting the method of induction of experimental diabetes mellitus by administering streptozotocin (STZ) in normal adult Wistar rats, this chapter aim is to establish the working protocol and to present the equipment used to monitor serum glucose level and serum total cholesterol level. Intraperitoneal injection of STZ (a synthetic antineoplastic agent) causes inflammation in the Langerhans islets with pancreatic -cells degeneration inducing experimental diabetes mellitus in about 2-4 days. 7. Evaluation of hypoglycemic effect of some vegetal species on streptozotocin-induced diabetes in rats The purpose of this study was to investigate the oral hypoglycemic effect of some vegetal products (Cynosbati fructus, Myrtilli folium, Myrtilli fructus, Phaseoli sine seminibus fructus, Trigonellae foenum-graeci semen, Violae tricoloris herba) on streptozotocin-induced diabetes in rats. The experimental groups were as follows: group 1, non-diabetic control rats; group 2, diabetic control rats untreated; group 1 and group 2 were orally administered distilled water daily using an intragastric tube; groups 3-8, diabetic rats treated (categorized by alphabetical order of the vegetal species administered) fed orally daily by an intragastric tube with powder (of each vegetal species used, categorized alphabetically) suspended in distilled water.

Statistical analysis: values are presented as means SD of 5 rats in each group. All the data were statistically evaluated by t-value two-sided test; the limit of statistical significance was set at p-level < 0.05. The treatment of diabetic animals with the vegetal species powder produced a significant decrease in blood glucose concentrations as compared with untreated diabetic control animals; the results indicated the potent anti-diabetic activity of Myrtilli folium (G4), Myrtilli fructus (G5), Phaseoli sine seminibus fructus (G6), Trigonellae foenum-graeci semen (G7) and slightly anti-diabetic activity of Violae tricoloris herba (G8) and Cynosbati fructus (G3). A significant decrease in total cholesterol was determined by the oral administration of the Trigonellae foenum-graeci semen powder (G7), followed by Cynosbati fructus powder (G3) and a slightly decrease was observed at animals treated orally with Myrtilli folium powder (G4). The results indicate the possibility that these vegetal species could be effective to prevent or retard the development of diabetes complications due to metabolic disorders. 8. Research on the hypoglycemic effect, cholesterol-lowering and antioxidant effect of plant species in rats with experimental streptozotocin-induced diabetes The aim of this study is the evaluation of hypoglycemic and cholesterol-lowering effect by determination of serum glucose level and serum total cholesterol level after oral administration of tinctures derived from plant products less studied in this respect (Syringae vulgaris flos f. lba, Syringae vulgaris flos f. violcea) in streptozotocin-induced diabetes in rats. It was also studied the antioxidant effect of the products listed, as well as some plant products known for their antidiabetic effect (Myrtilli folium, Phaseoli sine seminibus fructus) by determining the activity of antioxidant enzymes (superoxide dismutase SOD, glutathione peroxidase GPx, glutathione reductase - GR) and lipid peroxidation evaluated as level of reactive substances with tiobarbituric acid. Also, histological analysis was performed in order to correlate the effect of treatment on biochemical parameters of interest with changes in the liver and kidney tissue for the different study groups. The experimental groups were as follows: group 1, non-diabetic control rats; group 2, diabetic control rats untreated; group 1 and group 2 were orally administered distilled water daily using an intragastric tube; groups 3-6, diabetic rats treated (named according to the initial letters of the used products): G3 (SV Syringae vulgaris flos f. violcea), G4 (SA

Syringae vulgaris flos f. lba), G5 (MF Myrtilli folium) and G6 (PP Phaseoli pericarpium) fed orally daily by an intragastric tube with tinctures 20% suspended in distilled water. At the end of the six weeks of treatment, after 12 hours of fasting, animals were sacrificed and the collected blood was used for biochemical analysis. The liver and kidney tissue were harvested for histological analysis. Statistical analysis: values are presented as means SD of 5 rats in each group. All the data were statistically evaluated by t-value two-sided test; the limit of statistical significance was set at p-level < 0.05. The treatment of diabetic animals with tinctures from the studied plants significantly decreased glucose levels compared with untreated diabetic animals, the results confirming a potent antidiabetic activity for Myrtilli folium and Phaseoli pericarpium and a moderate antidiabetic activity for Syringae vulgaris flos f. violcea, respectively Syringae vulgaris flos f. lba. It was also observed a significant decrease in total cholesterol levels compared with untreated diabetic animals, the results showing a significant cholesterol-lowering activity when it was given Myrtilli folium, respectively Phaseoli pericarpium and a moderate reduction of total cholesterol in diabetic animals treated with tinctures of Syringae vulgaris flos f. violcea and Syringae vulgaris flos f. lba. The research has revealed an important antioxidant effect, with the increased of SOD, GPx and GR activity and the decreased of lipid peroxides after the treatment with tinctures of Myrtilli folium and Phaseoli pericarpium and a capacity of antioxidant enzymes stimulation and reducing lipid peroxidation for tinctures of Syringae vulgaris flos f. violcea and Syringae vulgaris flos f. lba. Regarding the histological analysis of pathological changes occurring in diabetic animals treated with tinctures from studied plant products (groups G3-G6), it is noted discrete changes witch confirms favorable development after six weeks of treatment and beneficial role of administration of these herbal remedies. We noted a correlation between changes in biochemical parameters determined and the degree of tissue deterioration.

FINAL CONCLUSIONS 1. Diabetes mellitus has become a major problem for the individual, medicine and society. The alarming find is that, when diagnosed type 2 diabetes, a high percentage of people already have chronic complications and/or morbid associations. 2. Diabetes mellitus is defined as a group of metabolic disorders characterized mainly by hyperglycemia induced by impaired insulin secretion, insulin resistance or both entities in varying proportions. 3. Besides oral agents and insulin therapy, phytotherapy is an alternative source that provides a range of natural resources with hypoglycemic effects, a range of plants providing raw materials recommended for people with diabetes. 4. Medicinal plants in different oral formulations were recommended to the diabetic patient, but the mechanisms for hypoglycemic activity still remained incompletely understood. 5. Among the most popular medicinal species with hypoglycemic effect we mentioned: Allium cepa, Allium sativum, Arctium lappa, Brassica sp., Centaurium umbellatum, Cynara cardunculus subsp. scolymus, Gentiana sp., Glycyrrhiza glabra, Morus sp., Phaseolus vulgaris, Rubus sp., Salvia officinalis, Taraxacum officinale, Trigonella foenum-graecum, Vaccinium myrtillus. 6. Plants with hypoglycemic activity from Romanias spontaneous flora, are mostly from families: Asteraceae, Cruciferae, Ericaceae, Fabaceae, Gentianaceae, Lamiaceae, Liliaceae, Moraceae, Polygonaceae, Rosaceae. 7. Following pharmacognostical research on obtaining and chemical and physical characterization of tinctures of some hypoglycemic plant products (Cynosbati fructus, Galegae herba, Myrtilli folium, Myrtilli fructus, Phaseoli pericarpium, Syringae vulgaris flos f. lba, Syringae vulgaris flos f. violcea, Trigonellae foenum-graeci semen, Violae tricoloris herba), their quality was determined according to Romanian Pharmacopoeia, 10th Edition and has conducted qualitative and quantitative analysis for flavonoids and phenyl-propane compounds. 8. Analysis tinctures revealed as active principles flavonoids and polyphenolic acids as active principles with a remarkable therapeutic potential and who are giving plant products hypoglycemic and antioxidant properties. 9. For the first time were characterized as such without further processing, both in terms of absorption peaks and pads in VIZ and specific

vibration in IR, tinctures obtained by simple percolation of the hypoglycemic plant products taken in study. 10. The preliminary study on hypoglycemic effect on plant species, experimental hyperglycemia was induced by subcutaneous administration of adrenaline in normal rat. From research conducted for Mori Folium powder that turns out to be a remedy with mild hypoglycemic activity, thereby urging the combination with other hypoglycemic addition of pharmacodynamic action. Robiniae pseudoacaciae folium and Violae tricoloris herba powders marginally reduced hyperglycemia in rats compared with control, but favoring a return of glucose values close to the reference. 11. The evaluation of Syringae vulgaris flos f. lba and Syringae vulgaris flos f. violcea tinctures on blood glucose levels when performed oral glucose tolerance test in normal rats showed improving response to exogenous glucose load. This pharmacodynamic effect was not shown so far from the studied plant products. 12. The study to asses the toxic potential after oral administration of tincture of Syringae vulgaris flos f. alba and Syringae vulgaris flos f. violcea by conducting in vivo experiments in normal rats showed that they are non-toxic products. 13. The research to investigate the oral hypoglycemic effect of some vegetal products (Cynosbati fructus, Myrtilli folium, Myrtilli fructus, Phaseoli sine seminibus fructus, Trigonellae foenum-graeci semen, Violae tricoloris herba) on streptozotocin-induced diabetes in rats proved that the treatment during four weeks of diabetic animals with the vegetal species powder produced a significant decrease in blood glucose concentrations as compared with untreated diabetic control animals. The results indicated the potent anti-diabetic activity of Myrtilli folium, Myrtilli fructus), Phaseoli sine seminibus fructus, Trigonellae foenum-graeci semen and slightly antidiabetic activity of Violae tricoloris herba and Cynosbati fructus. 14. A significant decrease in total cholesterol was determined by the oral administration of the Trigonellae foenum-graeci semen powder, followed by Cynosbati fructus powder and a slightly decrease was observed at animals treated orally with Myrtilli folium powder. 15. The results indicate the possibility that these vegetal species could be effective to prevent or retard the development of diabetes complications due to metabolic disorders. 16. In the research on the hypoglycemic effect, cholesterollowering and antioxidant effect of plant species in rats with experimental streptozotocin-induced diabetes we studied the effects after oral administration of tinctures derived from plant products less studied in this

respect (Syringae vulgaris flos f. lba, Syringae vulgaris flos f. violcea), as well as tinctures of some plant products known for their antidiabetic effect (Myrtilli folium, Phaseoli sine seminibus fructus). 17. The results after six weeks treatment of diabetic animals confirmed a potent antidiabetic activity for Myrtilli folium and Phaseoli pericarpium and a moderate antidiabetic activity for Syringae vulgaris flos f. violcea, respectively Syringae vulgaris flos f. alba and also a significant cholesterol-lowering activity when it was given Myrtilli folium, respectively Phaseoli pericarpium and a moderate reduction of total cholesterol in diabetic animals treated with tinctures of Syringae vulgaris flos f. violcea and Syringae vulgaris flos f. lba. 18. The research has revealed an important antioxidant effect, with the increased of SOD, GPx and GR activity and the decreased of lipid peroxides after the treatment with tinctures of Myrtilli folium and Phaseoli pericarpium and a capacity of antioxidant enzymes stimulation and reducing lipid peroxidation for tinctures of Syringae vulgaris flos f. violcea and Syringae vulgaris flos f. lba. 19. Regarding the histological analysis of pathological changes occurring in diabetic animals treated with tinctures from studied plant products, it is noted discrete changes wich confirms favorable development after six weeks of treatment and beneficial role of administration of these herbal remedies. We noted a correlation between changes in biochemical parameters determined and the degree of tissue deterioration. 20. The research on antioxidant status after treatment with powder and vegetal extracts in rats with experimentally streptozotocin-induced diabetes by determining the enzymatic activity of SOD, GPx and GR and the index of lipids peroxidation and also by correlating the changes of biochemical parameters of interest with changes in tissue liver and kidney, was not mentioned in the literature of our country, representing the originality of this thesis. 21. Research carried out shows that diabetic animals are subjected to oxidative stress and that oral administration of tinctures of plant products can reduce part imbalance between reactive oxygen species generation and antioxidant enzymes activity. Therefore, plant remedies are useful both in prevention and improvement of certain disease like diabetes, atherosclerosis and the complications caused by them. 22. For the first time was studied the effect of oral administration of tinctures of Syringae vulgaris flos f. violcea and Syringae vulgaris flos f. alba in streptozotocin-induced diabetic rats, results showing existing antidiabetic action and lowering serum total cholesterol, with a ability to stimulate antioxidant enzymes activity and reduce lipid peroxidation.

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CURRICULUM VITAE Name: BERBECARU-IOVAN First name: ANCA Birth date: 15.11.1970 Birth place: Craiova, Dolj Present: married, one child Address: Craiova, tefan cel Mare street, bloc 13, sc. A, apt. 11 Studies: High School of Mathematics and Physics Nicolae Blcescu, Craiova, Mathematics and Physics Profile, 1985-1989; Faculty of Medicine, General Medicine Specialization, University of Craiova, 1990-1996. Professional activity: Resident doctor in Emergency Medicine specialization, 1997-2000; Specialist doctor in 2000; currently enrolled in the training program in the second specialization Internal Medicine; Junior Assistant by competition 2000-2002, University Assistant by competition 2002-2004, Assistant Professor by competition 2004 present at the discipline of Pharmacology, Faculty of Pharmacy, University of Medicine and Pharmacy from Craiova. Active member in scientific societies: Member of Romanian Society of Pharmacology, Therapeutics and Clinical Toxicology. Member of the Society of Pharmaceutical Sciences from Romania. Member of the Society of Pharmacy History. Postgraduate courses: graduate of Department of Training Teacher (2006); graduate courses: Assistant Manager of Management Research improvement programme (2007); Euroson School, Guidelines in Gastroenterogical Ultrasound (2007); Theoretical and practical electrocardiography (2007); Liquid chromatography (2008); Pharmacotherapeutic education of patient (2008); Pharmacogenetics and Pharmacogenomics (2008); Insulin therapy in diabetes (2008). Scientific papers published in extenso 15 Scientific papers at national and international scientific meetings published in abstracts 34 Books and educational materials published (co-author) 3 Attendance at the National and International Congress of Clinical Pharmacology, Therapeutics and Toxicology 7 Attendance in other medical and pharmaceutical symposiums - 13 Foreign language: English, French.