Epothilones, a class of microtubule-targeting agents, are secondary metabolites of ... The study demonstrated that epothilone A and epothilone B had higher.
Rev. Med. Chir. Soc. Med. Nat., Iaşi – 2013 – vol. 117, no. 1
MEDICAL BIOENGINEERING
ORIGINAL PAPERS
CONTRIBUTIONS TO THE PHYTOCHEMICAL STUDY OF BIDENS TRIPARTITAE HERBA FROM ROMANIA I. TANNINS. M. Zagnat 1, Corina Cheptea 1, A. F. Şpac2 University of Medicine and Pharmacy “Grigore T. Popa” - Iasi 1. Faculty of Medical Bioengineering 2. Faculty of Pharmacy
CONTRIBUTIONS TO THE PHYTOCHEMICAL STUDY OF BIDENS TRIPARTITAE HERBA FROM ROMANIA. I.TANNINS (Abstract): Aim: To analyze qualitatively and quantitatively tannins in the native plant, collected during the whole vegetation period from different areas of the country, and in its different organs (flower, stem, leaf). Material and methods: For quantitative analysis, the plant product was extracted by repeated maceration (3 days) with 80% methanol. Proanthocyanidins in the extract were quantified by spectrophotometric methods. Results: condensed tannins were present while hydrolyzed tannins were absent. Chromatographic analysis showed that tannins spectrum is similar in all plant organs and in plants collected at different times throughout the vegetation period. The di fferences are only quantitative. The maximum amount of tannins was found during the flo wering stage (10.32%). Conclusions: In terms of tannin content, flowering is the best time to collect. However, collection throughout the whole vegetation period is acceptable. Keywords: MEDICINAL PLANTS, BIDENS TRIPARTITA L., TANNIN.
Bidenstripartita L. (family Asteraceae), usually named Burr marigold and Water agrimony, is an erect annual plant, growing 15 to 100 cm high. (1, 2, 3, 4, 5, 6). The plant is known and used since ancient times, especially for treatment of dermal diseases and wound healing (2). In popular medicine, it is orally administered as a diuretic, diaphoretic, febrifuge, antidiarrhoeal, antiallergic, anti-inflammatory, anthelminthic, choleretic and as a kidney tonic (1, 5, 6, 7). All these effects were confirmed by pharmacological tests or clinical studies. Moreover, good results have been reported in treating chronic dysentery, acute and chronic enteritis, ulcerative colitis, bladder and kidney problems
and some forms of cancer (1, 8). The chemical composition is not well studied. Some authors even specify this. In the last URSS pharmacopoeia the product is standardized according to polyholosides, but their presence does not account for all therapeutic properties of the plant. It is known that the dried aerial parts contain: flavones (luteolin, cynaroside), tannins, polyacetylenes, coumarins (umbelliferone, scopoletin and aesculetin), 0.05-0.11% essential oil (eugenol, ocimene and cosmene), 4.51-4.65% saccharides (arabinose, galactose, glucose, rhamnose and xylose), organic acids, carotene, vitamin C and microelements (manganese) (1, 3, 4, 8, 9). In the literature we found almost noth-
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ing about tannins in this plant. Considering the therapeutic properties of tannins (astringent, anti-inflammatory, antioxidant, anti-microbial and others) we can presume that they are extremely important components responsible for the numerous therapeutic uses of the product Bidenstripartitaeherba. Also unknown is the best time for collecting the plant material. Some authors recommend the period of flowering, others budding and/or flowering (1, 3), or just budding (5, 6, 9). Some of them admit the later collection, but only the side branches, with flower buds (10), others are very strict about insisting that the product collected after the beginning of flowering has no curative properties (4). For this reason, we decided to collect the native plant during the whole vegetation period in different regions of the country. Our aim wasto study tannins qualitatively and quantitatively in the collected plant material, and their content in different plant organs (flower, stem, leaf). MATERIAL AND METHODS The vegetative material originated from Iasi area and Ceahlau massif and was collected in the summer of year 2012, from late June to early October, for approximately equal time periods (10-15 days). The collection was made at different times during the afternoon. The plant was identified based on macro- and microscopic characters (1, 3) and dried in our laboratory. For the qualitative analysis the air-dried samples of raw plant material were cut into 2-3 mm particles and then extracted using water: 5 g of dried herb was extracted with 100.0 ml boiling water on water bath for 15-20 min. The obtained extract was filtered through filter paper and was used to
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carry out tannins specific qualitative reactions (11, 12). For the TLC analysis we chose some more representative samples. We prepared a 1:10 ethanolic extract for each analyzed sample. The extracts were subjected to chromatographic separation. From each sample 10 μl of extractive solution was spotted on the chromatographic plate. The working conditions are: - stationary phase - Silica gel G F 254precoated TLC plates (Merck, Germany); - mobile phase: n-buthanol / glacial acetic acid / water (40 / 12 / 28, v/v/v); - detection: spaying with 1% vanillin in concentrated HCl (12). For quantitative analysis, 2 g of crushed plant material taken from each sample has been extracted by repeated maceration method (3 days) with 25 ml of 80% methanol. For comparison, a sample extraction was performed by repeated maceration and stirring (magnetic stirrer).The crushed product was extracted with 3 equal portions of 80% methanol in 3 one-hour cycles. Hydrolysis of proanthocyanidin was performed as described by Porter et al. (13, 14). The iron reagent was a 2% (w/v) solution of NH 4Fe(SO 4)2·12H2O in 2M HCl. 0.5 ml of analyzed extract, 3.0 ml of nbutanol solution and conc. HCl (95:5 v/v) and 0.1 ml of iron reagent were added to the glass tube sealed with a cap. The reaction mixture was then heated for 1 h at 950C on water bath and then red at 550 nm in a 10 mm lath-length glass cell. The results were expressed as milligrams of cyanidin equivalents per gram of extract using ε = 17,360 L mol-1 cm-1 and MW = 287.24 (15). RESULTS The results of qualitative reactions for the
Contributions to the phytochemical study of Bidens tripartitae herba from Romania. I. tannins.
tannins: with lead acetate-precipitate (tannins), with basic zinc acetate-green precipitate (tannins), with FeCl3soln-green precipitate (condensed tannins), with iron alum-dark
green precipitate (condensed tannins), with styassny reagent-red precipitate (condensed tannins), filtrate gave no precipitate with iron alum (no hydrolysable tannins).
Fig. 1. Chromatogram for tannins during the vegetation period. Sample numbers correspond to those in tab. I. TABLE I Tannins concentration depending on collection time Observations
Concentration %
No sample
Time of collection
Location of collection
1
17.06
Ciric area
6.80
2
27.06
-„-
3.81
3
11.07
-„-
7.03
4
23.07
-„-
Water deficiency
5.02
5
-„-
-„-
Optimal growth conditions
3.91
6
-„-
-„-
Heavily pigmented plants
4.75
7
16.08
-„-
6.52
8
21.08
Bahlui
4.21
9
22.08
Ceahlau
6.59
10
11.09
Ciric area
Flowering plants
10.32
11
-„-
-„-
Plants with fruits
6.17
12
2.10
Osoi
Only leaves
4.11
13
27.06
Ciric area
Maceration with agitation
3.98
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12
C,%
10
10 8
3
1
6 4
4 2
9
7 6 5
11
8
12 13
2 0
No. sample
Fig. 2. Tannins concentration depending on collection time. TABLE III Tannins concentration in the organs of B. tripartitaeherba. No 1 2 3 4 Plant organ flower stem leaf herba Conc., % 2.57 1.46 8.91 4.98 C,%
; 3; 8,91
; 4; 4,98 ; 1; 2,57 ; 2; 1,46 No sample
Fig. 3.Tannins concentration in the organs of B. tripartitaeherba. DISCUSSION From the results of qualitative reactions (tab.I) we conclude that in this plant product the condensed tannins are present while hydrolysed tannins are missing. This confirms the results of thin layer chromatography. Moreover, chromatographic analysis showed that tannins spectrum is similar in
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all plant organs and in plants collected at different times throughout the vegetation season. The differences are only quantitative (fig. 2,3). Samples 4, 5 and 6 were collected on the same day (afternoon of July 23), from the same area but in locations with different environmental conditions. Sample 4 was collected from the banks of dried up pond. Plants were small size (about 25 cm), highly pigmented and at an advanced stage of development. Many specimens were in fruiting stage (we did not collect them because of indications in the literature on optimal collection period). Samples 5 and 6 were collected from an area intensively irrigated by a creek. Sample 5 had a reduced pigmentation, while sample 6 was heavily pigmented. The determinations showed that sample 5 is less rich in tannins than the other two samples. Sample 7 also included some plants in budding and flowering stage. Samples 8 and 9 were collected on two consecutive days. Sample 8 was collected from a wetland, partially shaded on the banks of Bahluiriver (altitude less than 100 m above sea level). Sample 9 was collected from Ceahlau massif (approx. altitude 800 m). Due to drastic reduction of flow and stream channels narrowing, some plants grew on rocky soil, exposed to direct sun light and away from water. Sample 9 was obviously richer in tannins. This is probably accounted for by altitude, but other factors that might influence the biosynthesis of tannins should not be ignored. Samples 10 and 11 were collected on the same day and location. Sample 10 included only flowering plants. Sample 11 included only the plants with fruits. Sample 10 contained the highest amount of tannins of all samples. Although Sample 11 contained less tannins, it exceeded the average for all study samples. This finding allows us
Contributions to the phytochemical study of Bidens tripartitae herba from Romania. I. tannins.
to conclude that the opinion according to which after flowering the plant should not be collected is unfounded, at least in terms of tannin concentration. Sample 12 included only leaves, the other parts being removed because most of the collected plants had lignified stems. These are not usually used for the preparation of a drug. Results fit, however, to the levels of evidence with the lowest concentrations. So it is possible to even use them. Sample 13 is similar to sample 2, but extracted by shaking with slight heating magnetic stirrer. The results are very close. We chose the classic repeated maceration because it was easier to implement. The differences in concentration might be due to the fact that we collected samples from different areas around Iasi with different habitat conditions. It is known, for example, that tannins biogenesis is dependent on the intensity of photosynthesis (11). This happened because the populations of previously collected plants were destroyed by human activities. CONCLUSIONS The Bidenstripartitaeherba plant product contains condensed tannins.
The average concentration throughout the vegetation period is 5.77%. Tannins are present in all plant organs. The highest amount was found in the leaves, the lowest in the stem. During the vegetation period the highest amount of tannins was at flowering. In terms of tannin content flowering is the best time to collect. Somewhat lower values were found throughout the vegetation period, fruiting period included. Thus, the collection throughout the vegetation period may be acceptable. The obtained results allow us to conclude that water deficit increases the concentration of tannins. We also found that heavily pigmented specimens contain a larger amount of tannins. Plants collected at high altitudes were also rich in tannins, but they were collected from drier places and were intensely pigmented. ACKNOWLEDGEMENTS This research was financially supported by the Internal research grant of University of Medicine and Pharmacy “Grigore T. Popa”-Iasi Nr. 28209/16/12/2011. Special thanks to Mrs. Prof. Dr. AncaMiron for providing the ferric reagent and her help.
REFERENCES 1. *** WHO monographs on medicinal plants commonly used in the Newly Independent States (NIS). (Rus.). World Health Organization, 2010. 2. Gubanov IA, Kiseleva KV, Novikov VS, Tihomirov VN. Illustrated plants determinator of Middle Russia(Rus.). Moscow: Company of scientific publishing houses КМК, Technology Research Institute, vol. 3, 2004. 3. Muravieva DA, Samilina IA, Yakovlev GP. Pharmacognosy (Rus.). Moscow: Medicine, 2002. 4. Popov VI, Shapiro DK, Danusevichi IK. Medicinal plants (Rus.). 2-nd Edition, Minsk: Ed. Flame, 1990. 5. Poludenniy LV, Sotnik VF, Halaptsev EE. Aromatic and medicinal plants (Rus.). Moscow: Ed. Spike, 1979.
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6. Turova AD, Sapojnikova EN. Medicinal plants of the USSR and their application.(Rus.). 4-th Edition, Moscow: Medicine, 1984. 7. Maznev NI. The encyclopedia of medicinal plants (Rus.). Moscow: Ed. Martin, 2004. 8. Pozharitskaya ON et al. Anti-inflammatory activity of a HPLC-fingerprinted aqueous infusion of aerial part of Bidenstripartita L. Phytomedicine 2010; 17: 463-468 9. Shreter AI. Medicinal Flora of Soviet Far East (Rus.). Moscow: Medicine, 1979. 10. Gamerman AF.Wild medicinal plants. (Rus.). Medicine, Moscow, 1976. 11. Ciulei I, Grigorescu Em, Stănescu U. Medicinal plants, Phytochemistry and phytotherapy (Rom.).Bucharest: Medical Publishing House, V. 1, 1993. 12. Ladygina EY, et all. The Chemical Analysis of Medicinal Plants. Moscow: The Higher School, 1983 13. Porter JL, Hrstich LN, Chan BG. The conversion of procyanidin and prodelphinidin to cyanidin and delphinidin, Phytochemistry1986; 25: 223-230. 14. Apetrei CL, Boz I, Cenusa E, Ionescu A, Miron A. Pinicembraeconi: studiihisto-anatomicesichimice, evaluareaactiuniiantioxidante, Rev. Med.Chir. Soc. Med. Nat., Iasi 2012; 116 (2): 635-641. 15. Qa'dan F, Petereit F, Mansoor K, Nahrsted A. Antioxidant oligomericproanthocyanidins from Cistussalvifolius. Natural Product Research 2006; 20: 1216-1224.
NOUTĂȚI EPOTHILONE A AND EPOTHILONE B, PROMISING SUBSTANCES FOR TREATMENT OF HUMAN OVARIAN CANCER Epothilones, a class of microtubule-targeting agents, are secondary metabolites of myxobacteria. A new study on epothilone A and epothilone B showed that they could be good candidates for ovarian cancer treatment. Epothilones have a taxane-like mechanism of action, but are more effective against P-glycoprotein overexpressing cancer cells. The effects of epothilone A and epothilone B, obtained from myxobacterium Sorangium cellulosum, were investigated on SKOV-3 human ovarian carcinoma cell line and the results were compared with those of paclitaxel. The study demonstrated that epothilone A and epothilone B had higher cytotoxic effects (IC50 = 20.4 nM and 57.5 nM, respectively) than paclitaxel (IC50 = 126.2 nM) in MTT assay. In addition, they induced apoptosis in ovarian carcinoma cell line through reactive oxygen species (ROS) production. This could be considered one of the mechanisms involved in epothilones cytotoxicity. The oxidative stress caused by ROS d etermine disruption of mitochondrial membrane function and has as consequence the indu ction of apoptosis. The results showed that epothilone A and epothilone B caused greater d epolarization of the mitochondrial membrane than paclitaxel. Preliminary data of this study indicate that epothilone A and epothilone B can be used in the near future in ovarian cancer treatment with better results than those of paclitaxel (Rogalska A, Marczak A, Gajek A et al. Induction of apoptosis in human ovarian cancer cells by new anticancer compounds, epoth ilone A and B. Toxicol in Vitro 2013; 27: 239-249). Cristina Lungu
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