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B. Kdzia, T. Bobkiewicz-Kozowska, M. Furmanowa. P. Mikoajczak, E. Hoderna-Kdzia, I. Okulicz-Kozaryn, ...

Studies on the biological properties of extracts from Centella asiatica (L.) Urban herb

BOGDAN KDZIA1*, TERESA BOBKIEWICZ-KOZOWSKA2, MIROSAWA FURMANOWA3, PRZEMYSAW MIKOAJCZAK2, ELBIETA HODERNA-KDZIA1, IRENA OKULICZ-KOZARYN2,JOANNA WJCIK1, JOANNA GUZEWSKA3, WALDEMAR BUCHWALD1, ALINA MCISZ1, PRZEMYSAW M. MROZIKIEWICZ1

1Research Institute of Medicinal Plants, ul. Libelta 27, 61-707 Pozna, Poland2 Department of Pharmacology, University of Medical Sciences,ul. Rokietnicka 5a, 60-806 Pozna, Poland3Department of Biology and Pharmaceutical Botany, Medical University, ul. Banacha 1, 02-097 Warszawa, Poland

*corresponding author: phone: +4861 6659540 ext. 11, fax: +4861 6659551, e-mail: [email protected]

S u m m a r y

The aqueous-ethanol extracts (30% ethanol) from Centella asiatica have a stronger antibiotic activity compared to other extracts. In comparison to tetracycline, the antibiotic activity of above extracts can be determined as a poor. The aqueous-ethanol extracts (60% ethanol) showed a stronger antioxidative activity compared to other extracts. In comparison to chlorogenic acid the antioxidative activity of above extracts is poor. There was found no difference in antibiotic and antioxidative activity between extracts obtained from hydroponic and field cultivation.On the other hand, it was found that aqueous-ethanol extract from the herb of Centella asiatica shows the bacteriostatic activity to all Gram-positive and Gram-negative bacteria isolated from wounds of different etiology (MIC, 1040 mg/ml). This extract did not show any irritative and mutagenic activity according to OECD directives. The mentioned extract applied topically in form of 1% cream showed tendency to reduce oedema induced by car-rageen after 3 h from application (ca. 12%).We conclude that the topically applied preparation (cream or gel) containing 5% of inves-tigated aqueous extract from herb of Centella asiatica (50 mg/g of preparation) can be used for healing of infected wounds in hospitalized patients.

Key words: Centella asiatica extracts, antibiotic, antioxidative, mutagenic, irritative and antin-flammatory activity.

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INTRODUCTION

Centella asiatica (L.) Urban (syn. Hydrocotyle asiatica L.) is a plant from Umbelliferae family occurs in India, China, Indonesia and in hot climate zone. A crude drug is a herb containing a triterpene compounds complex.

In the Research Institute of Medicinal Plants the trials on hydroponic and field cultivation of a plant material obtained from in vitro cultures (Department of Biology and Pharmaceutical Botany, Medical University of Warsaw) have been carried out. The crude drugs obtained in Polish edaphic-climatic conditions have been estimated in phytochemical aspect. The aqueous and aqueous-ethanol ex-tracts obtained from those crude drugs were analyzed [1, 2]. In both Institutes also biological studies including the influence of extract from Centella asiatica on the chemokinetic activity of mice splenic lymphocytes and the influence on cutaneous angiogenesis induced by L-1 sacroma cells in mice were performed [3, 4].

Studies performed in last years show that extracts obtained from Centella asia-tica (L.) Urban appear to have many valuable biological properties, especially wo-und healing connected with antimicrobial [5-8], antiinflammatory, antioxidative and immunostimulating activity of extracts from this plant [5, 8-11]. For the above properties are responsible triterpene derivates including asiatic acid and asiatico-side, phenolic compounds with flavonoids and volatile oil. Moreover, the antitu-mor, anxiolytic and psychoneurological activity of extracts from Centella asiatica is emphasized [6, 8, 10-12]. The triterpene fraction from this plant is used in chronic venous insufficiency and in microangiopathy [14].

The different properties of Centella asiatica extracts caused that many medical products are used in Polish and European markets. In Poland there are known: Madecassol and Analen (ointments), Alveo (liquid), Varixinal (tablets) and Cicatri-dine (globules) [3].

The aim of our study was to estimate antibiotic (antimicrobial), antioxidative and antiinflammatory as well as mutagenic and locally irritative activity of Centella asiatica extracts of native origin in aspect of preparation for wound healing.

MATERIAL AND METHODS

Investigated extracts

In the studies there were used 6 dried extracts obtained from the herb of Cen-tella asiatica (L.) Urban. The crude drug was from hydroponic and field cultivations conducted in the Research Institute of Medicinal Plants in Pozna. The studies were performed on aqueous extracts (C1 and C4), aqueous-alcoholic extracts ob-tained with 30% ethanol (C2 and C5) and aqueous-alcoholic extracts obtained with 60% ethanol (C3 and C6).


Antibiotic activity

The studies were carried out with use of the dilution method in fluid medium [11]. The extracts were dissolved in DSMO in concentration of 100 mg/ml. Then the dilution in fluid growth medium Antibiotic Medium I (Difco) in concentration of 101000 g was prepared. To the prepared dilutions in a volume of 1 ml there was added 0.1 ml of Staphylococcus aureus FDA 209P, incubated for 24 hours inclu-ding 105 cells in 1 ml. After incubation at the temperature of 37oC the minimal concentration of investigated extracts inhibiting development of standard strain (MIC - Minimal Inhibitory Concentration) was determined. A control test was per-formed with tetracycline (Merck).

Antioxidant properties

The studies were performed in vitro. The test with DPPH (2,2-diphenyl-1-picryl-hydrazyl) was used, according to Hatano et al. [16]. The extracts were dissolved in DMSO in concentration of 100 mg/ml and then diluted in distilled water. As a source of free radicals 0.004% DPPH solution (Sigma) in ethanol was used. DPPH solution in amount of 2.5 ml was placed in a glass tubes with addition of 0.1 ml of investigated extract. The components were mixed and stored for 30 min. in a dark place. After this time the absorbance of investigated sample by a wave length =517 nm was measured compared with a blank test (2,5 ml of DPPH solution + 0.1 ml of DMSO).

Then the minimal concentration of extract in mg/ml characterized by a high rate of free radicals scavening (color reaction) was determined. The reference substance was chlorogenic acid (Merck).

The activity of aqueous extract on bacteria isolated from wounds

In the studies the aqueous-ethanol extract from the herb of Centella asiatica (field cultivation) in a dried form (C1) was used. The studies included 21 bacteria strains isolated from the wounds of different etiology from patients of Public Clinical Hospital No 5 in Pozna. From among investigated strains, 13 were Gram-positive and 8 Gram-negative. The studies were conducted with use of method of series dilution test in a fluid medium [15]. Extract was dissolved in DMSO in concentration of 500 mg/ml. Each 1 ml of adequate dilutions of a basic solution were introduced into Petri dishes of 9 cm diameter and poured with a fluid Mu-eller-Hinton Agar medium (Difco) in amount of 10 ml. After 24 h incubation at temperature 37oC in Mueller-Hinton medium (Mueller-Hinton Broth, Merck) the cultures of investigated bacteria were dissolved to obtain 104105 bacteria cells in 1 ml. The prepared dilutions of bacteria cultivations were inoculated by a score method on the surface of prepared plates containing 550 mg of investigated

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extract. The plates were incubated at the temperature of 370C and after 24 h the minimal inhibitory concentration (MIC) of extract inhibiting development of inve-stigated strains was determined.

Mutagenic activity

In the studies the aqueous-ethanol extract obtained from the herb of Centella asiatica (C1) was used. In experience back mutation the Salmonella typhimurium Ames test (17) was used. The histidine dependent Salmonella typhimurium TA 1535 (ATCC 29629) strain with reversion leading to forming of strain independent from histidine presence is inducted by defined mutagenic substances. Natrium azide was used as a mutagenic substance.

The investigated extracts were dissolved in DMSO in amount of 100 mg/ml and then diluted in the same solvent. To Ehrlenmayer flask containing 10 ml of fluid and cooled to 50oC top layer medium (medium B composed of 60 mg agar, natrium chloride 50 mg, distilled water 10 ml) following substrates in the amount of 0,1 ml were added: mixture of histidine and biotine, natrium azide, solution of investigated extract and diluted standard strain of S. typhimurium (cultivated 24 h, diluted in proportion of 1:10 in nutrient medium).

Then 1.5 ml of medium B with adequate substrates was placed on the surface of 10 ml of solid medium of lower layer (medium A containing 150 mg of agar, 0.2 ml of VBME salts (50 x) 0,2 ml, 0.5 ml of 40% glucose and 9.3 ml of distilled water) and was distributed uniformly on the plate surface.

The mixture of histidine and biotine was composed of 96 mg of L-histidine hydrochloride (Merck), 124 mg of D-biotine (Sigma) and 10 ml of distilled water. The mixture of VBME salts (50 x) was composed of magnesium sulphate x 7H20 1 g, citric acid x 1H20 10 g, acid anhydrous potassium phosphate 50 g, sodium am-monium phosphate x 4H20 17.5 g, distilled water 67 ml. Natrium azide was used in form of aqueous solution in concentration of 1 mg/ml.

After preparation plates were incubated at a temperature of 37oC for 72 h, pro-tected from drying. Then number of colonies of Salmonella typhimurium grown on the plates was determined and studies results were interpreted.

If in the plates containing 10.0 mg, 1.0 mg and 0.1 mg of investigated extracts were the same number of Salmonella typhimurium colonies as by growth control occurred: (below 100 colonies), it was assumed that the investigated extract did not show any mutagenic activity.

Irritative activity

The studies were performed on male rabbits (weight ca. 3.0 kg) housed inside a rooms with controlled temperature 202oC (humidity 65%) and 12 h light-dark cycle (light 7.0019.00 h), with free access to water and vegetables food (beets, carrots, rutabaga and hay).


The subject of studies was aqueous-ethanol extract from the herb of Centella asiatica in dried form (C2), applied topically as a 1% cream or 1% of aqueous sus-pension in amount equal to 5 mg of extract.

The estimation of the acute irritative activity was performed according to OECD directives for the chemical studies [18]. The study was conducted on 2 rabbits with removed pelage on the places prepared to apply investigated preparation. On the day of investigation 2 compresses with adequate vehiculum were applied. The first estimation of irritative activity was performed after 1 h. Each compress was removed from right and left side of animals spine. The other compresses were removed after 4 hours. The observation of animals skin was conducted after 12, 24 and 72 hours after the compresses removal, according to OECD directives.

Antiinflammatory activity

The studies were performed on the Wistar rats (body weight 180220 g) with a free access to water and standard food Labofeed B (LSM, Kcynia). The other conditions were the same as in the case of rabbits. In the studies was used aque-ous ethanol-extract from the herb of Centella asiatica (field cultivation) in dried form (C2). The estimation of antiinflammatory activity of studied substances was carried out using a plethysmometer (Ugo Basile), according to Choudhary et al. method [19].

The electrolytic fluid (solution of chloride of I-valency alkali metal in concen-tration of ca. 6 mmol/l) was prepared due to producers recommendations. One liter of fluid contained: 0.351 g NaCl, 3 ml of agent reducing the surface tension (Desprej, Biochemie) and distilled water.

For the measuring the rat paw was dipped on the same early determined depth in measuring chamber. After 4 s the result was read out.

The amount of fluid in chamber was complemented after each measuring to the zero level. Before measuring the paws were cleaned and the fluid changed after each animal for the protection against contamination of electrolytic fluid.

The inflammation was induced by the injection of 1% carrageenan solution (Car-rageenan Lambda, Sigma) into subplantar area of the left paw. The same amount of physiological salt solution NaCl was injected into the right paw [19]. The investiga-ted preparation in amount of 5 mg as a 1% cream was applied topically immediately after carrageenan application, putting than sterile dressing for close contact of inve-stigated preparation with hind left paw [20]. The right hind paw received placebo in this same way. After 1.5 and 3.0 h the antiinflammatory activity was measured.

As a reference compound indometacine in amount of 10 mg/kg s.c. (imme-diately after carrageenan application) was applied. Rats were anesthetized with thiopental sodium (60 mg/kg i.p.).

For the description of results formula of difference in change of volume of right hind paw and left hind paw after induction of inflammatory was used:

G=(Lt-Lo)-(Pt-Po),

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where: G value expressing difference in the change of volumen of right (Pt) and left (Lt) hind paw after 1.5 or 3.0 h on induction of inflammatory compared to output value]:, Lo start volume of left hind paw, P0 start volume of right hind paw.

All factors disturbing results were reduced (noise, presence of people or other animals). All values were expressed as an arithmetic mean SEM (n = number of rats included to each analysis). The statistic comparison was performed with use of the variance analysis (ANOVA) and test of minimal significant difference (LSD).

RESULTS

The results presented in Table 1 show that extracts C2 and C5 (aqueous-alco-hol extracts obtained with 30% ethanol) inhibited the development of standard strain Staphylococcus aureus FDA 209 P in concentration of 1000 g/ml. Other in-vestigated extracts inhibited the growth of standard strain below this value. As a comparison, tetracycline inhibited the growth of standard strain in concentration of 0.1 g/ml.

Ta b l e 1 .

Antibiotic activity of Centella asiatica herb extracts.

investigated extracts MIC (g/ml)C1 >1000C2 1000C3 >1000 C4 >1000C5 1000C6 >1000

tetracycline 0.1

The antioxidative activity of extracts from herb Centella asiatica is presented in Table 2. The studies results show that extracts C3 and C6 have a ten times stron-ger antioxidative activity in comparison to other extracts. For example, chloroge-nic acid was active in concentration of 0.01 g/ml.

Ta b l e 2 .

Antioxidative activity of herb Centella asiatica extracts.

investigated extracts MIC (g/ml)C1 10C2 10C3 1C4 10C5 10C6 1

chlorogenic acid 0.01


The activity of aqueous extract from herb Centella asiatica on Gram-positive and Gram-negative bacteria isolated from wounds are presented in Table 3. The ob-tained results indicate that Gram-positive bacteria are inhibited by a little higher concentration of extract from Centella asiatica (MIC mean=30 mg/ml) in compa-rison to Gram-negative bacteria (MIC mean=23 mg/ml). The highest resistance (MIC 2540 mg/ml) showed the strains of Staphylococcus aureus and Corynebacte-rium jejkeum (MIC=35 mg/ml). Other Gram-positive and all Gram-negative bacteria isolated from wounds were inhibited by this extract in the range concentration MIC=1025 mg/ml. It is very interesting, because usually Gram-negative bacteria like: Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa are characte-rized by repeatedly higher resistance to preparations of plant origin.

Ta b l e 3 .

Activity of aqueous-ethanol extract from herb Centella asiatica (C2) on Gram-positive and Gram-negative bacteria isolated from wounds.

strain place of isolation MIC(mg/ml)Gram-positive bacteria

1. Staphylococcus aureus decubitus 30.02. Staphylococcus aureus wound in anal area 35.0 3. Staphylococcus aureus frontal cranium absces 35.04. Staphylococcus aureus wound on ear concha 25.05. Staphylococcus aureus decubitus 35.06. Staphylococcus aureus perirectal fistula 40.07. Staphylococcus aureus burn wound 35.08. Staphylococcus epidermidis acne 15.09. Streptococcus pyogenes perirectal fistula 10.010. Enterococcus faecalis decubitus 25.0

11. Enterococcus faecalis wound 20.012. Enterococcus faecalis purulent by catheter outlet 10.013. Corynebacterium jejkeum wound after cardiosugery 35.0

Gram-negative bacteria 25.014. Escherichia coli wound after cardiosugery 20.015. Escherichia coli decubitus 25.016. Escherichia coli wound after cardiosugery 25.017. Escherichia coli perirectal fistula 25.018. Escherichia coli perirectal fistula 25.019. Escherichia coli purulent by catheter outlet 25.020. Klebsiella pneumoniae wound after cardiosugery 15.021. Pseudomonas aeruginosa decubitus

The results of studies on mutagenic activity of aqueous-ethanol extract from Centella asiatica are presented in Table 4. They show that investigated extract were not mutagenic active. Such activity exhibited natrium azide, a standard mutagenic substance.

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Ta b l e 4 .

Studies on mutagenic activity of aqueous-ethanol extract from Centella asistica (C1).

name of platesubstrates added to surface medium B

number of colonies of S.typhimurium on the plate

1. growth control of S. typhimurium I, IV 10003. investigated extract from C. asiatica (10 mg/plate) I, III.IV


3. No difference has been found between extracts obtained from hydroponic and field cultivation in aspect of antibiotic and antioxidative activity.

4. It was found that the aqueous-ethanol extract from the herb of Centella asiatica (C2) shows the bacteriostatic activity to all Gram-positive and Gram-negative bacteria isolated from wounds of different etiology (MIC 10-40 mg/ml).

5. The aqueous-ethanol extract from the herb of Centella asiatica (C2) did not shows any irritative activity according to OECD directives.

6. The aqueous-ethanol extract form Centella asiatica (C2) applied topically in form of 1% cream showed tendency to reduce oedema induced by carrageen after 3 h from application of preparation (ca. 12%).

7. It could be stated that the topically applied preparation (cream or gel) con-taining 5% of investigated aqueous-ethanol extract from herb of Centella asia-tica (50 mg/g of preparation) can be used for healing of infected wounds by hospitalized patients.

ACKNOWLEDGEMENTS

This study was supported by grant No PBZ-KBN-092/PO5/2003: Research on new sources of natural products with biological activity: antimicrobial, antiinflammatory, antioxidative and cytostatic obtained from selected plants species cultivated in vivo and in vitro using biotechnological methods.

REFERENCES

1. Buchwald W, Forycka A, Furmanowa M, Mcisz A, Mielcarek S, Mrozikiewicz PM. Uprawy prbne Centella asiatica (L.) Urban w Polsce. Herba Pol 2006; 52(3):67.

2. Mielcarek S, Przybylak JK, Buchwald W, Furmanowa M, Krajewska-Patan A, Mcisz A, Mrozikiewicz PM. Centella asiatica uprawiana w Polsce badania fitochemiczne metod RP-HPLC-DAD. Herba Pol 2006; 52(3):79.

3. Guzewska J, Skopiska-Rewska E, Mcisz A, Buchwald W, Biaas-Chromiec B, Filewska M, Furmanowa M. In vivo stimulatory effect of Centella asiatica extract on in vitro chemokinetic activity of murine spleen cells. Proceedings of the conference: Naturalne i syntetyczne modulatory odpowiedzi immunologicznej i angiogenezy, Jurata, czerwiec 2006:26.

4. Guzewska J, Skopiska-Rewska E, Mcisz A, Buchwald W, Biaas-Chromiec B, Filewska M, Furmanowa M. Selected biological activities of Centella asiatica extracts. 5th International Symposium on Chromatography of Natural Products (ISCNP), Lublin, 1922 June 2006:105.

5. Vogel HG, De Souza NJ, D`Sa A. Effect of terpenoids isolated from Centella asiatica on granuloma tissue. Acta Ther 1990; 16:285-98.

6. Brinkhaus B, Lindner M, Schuppan D, Hahn EG. Chemical, pharmacological and clinical profile of the East Asian medical plant Centella asiatica. Phytomed 2000; 7:427-48.

7. Sampson JH, Raman A, Karlsen G, Navsaira H, Leigh IM. In vitro keratinocite antiproliferant effect of Centella asiatica extract and triterpenoid saponins. Phytomed 2001; 8:230-5.

8. Berege L. Centella asiatica: a revive. Aust J Med Herbalism 2004; 16(1):15-27.9. Loiseau A, Thron E, Buche P, Sirvent A, Girard F. Evidencing the antimicrobial properties of Centella

asiatica. Euro Cosmet 2002; 4:20-2.10. Zainol MK, Abd-Hamid A, Yusof S, Muse R. Antioxidative activity and total phenolic compounds of leaf,

root and petiole of four accessions of Centella asiatica (L.) Urban. Food Chem 2003; 81:575-81.

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11. Centella asiatica selected triterpenes. Indena. http://www/indena.it. 12. Minija J, Thoppil JE. Antimicrobial activity of Centella asiatica (L.) Urb. essential oil. Indian Perfumer

2003: 47:179-81.13. Howes MJR, Houghton PJ. Plants used in Chinese and Indian traditional medicine for improvement of

memory and cognitive function. Pharm Biochem Behavior 2003; 75:513-27.14. De Sanctis MT, Belcaro G, Incandela L, Cesarone MR, Griffin M, Ippolito E, Cacchio M. Treatment of

edema and increased capillary filtration in venous hypertension with total triterpenic fraction of Centella asiatica: a clinical, prospective, placebo-controlled, randomized, dose-ranging trial. Angiology 2001; 52(Suppl. 2):S55-9.

15. Kavanagh F. Analytical microbiology. Academic Press, New York-London 1963:249-379.16. Hatano T, Kagawa H, Yasuhara T, Okuda T. Two new flavonoids and other constituents in licorice root:

Their relative astringency and radical scavenging effects. Chem Pharm Bull 1988; 36:1010-17. 17. Maron OM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res 1983; 113:173-

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BADANIA NAD WACIWOCIAMI BIOLOGICZNYMI WYCIGW Z ZIELA CENTELLA ASIATICA (L.) URBAN

BOGDAN KDZIA1*, TERESA BOBKIEWICZ-KOZOWSKA2, MIROSAWA FURMANOWA3, PRZEMYSAW MIKOAJCZAK2, ELBIETA HODERNA-KDZIA1, IRENA OKULICZ-KOZARYN2,JOANNA WJCIK1, JOANNA GUZEWSKA3, WALDEMAR BUCHWALD1, ALINA MCISZ1, PRZEMYSAW M. MROZIKIEWICZ1

1Instytut Rolin i Przetworw Zielarskich, Libelta 27, 61-707 Pozna2 Katedra i Zakad Farmakologii, Uniwersytet Medyczny, Rokietnicka 5a, 60-806 Pozna 3 Katedra i Zakad Biologii i Botaniki Farmaceutycznej, Akademia Medyczna, Banacha 1, 02-097 Warszawa

*autor, do ktrego naley kierowa korespondencj: tel.: +4861 6659540 w. 11, faks: +4861 6659551, e-mail: [email protected]


S t r e s z c z e n i e

Wycigi wodno-alkoholowe (30% etanolu) z ziela Centella asiatica odznaczay si sil-niejszym dziaaniem antybiotycznym w porwnaniu z pozostaymi wycigami. Dziaanie to mona okreli na poziomie sabej aktywnoci przeciwdrobnoustrojowej w odniesieniu do tetracykliny. Wycigi wodno-alkoholowe (60% etanolu) wykazyway silniejsze dziaanie przeciwutleniajce w porwnaniu z pozostaymi wycigami. Dziaanie to mona okreli jako sabe w odniesieniu do kwasu chlorogenowego. Nie stwierdzono rnic w dziaaniu antybiotycznym i przeciwutleniajcym wycigw z ziela Centella asiatica pochodzcego z upraw hydroponicznych i gruntowych.Stwierdzono, e wodno-alkoholowy wycig z ziela Centella asiatica wykazuje dziaanie bak-teriostatyczne wobec wszystkich bakterii Gram-dodatnich i Gram-ujemnych wyizolowanych z ran o rnej etiologii (MIC w granicach 1040 mg/ml).Wycig ten nie wykazywa dziaania mutagennego w mikrobiologicznym tecie Amesa. Podawany w formie 1% kremu i 1% za-wiesiny wodnej nie wykazywa dziaania dranicego wg wytycznych OECD. Omawiany wycig, podawany miejscowo w formie 1% kremu, po jednorazowej aplikacji wykazywa tendencj do zmniejszania powstajcego obrzku wywoanego karagenin po 3 godz. od podania preparatu (o ok. 12%).Na podstawie przeprowadzonych bada mona wnioskowa, e preparat miejscowy (w postaci kremu lub elu), zawierajcy w swoim skadzie 5% badanego wycigu wodno-alkoholowego z ziela Centella asiatica (50 mg/g preparatu), moe znale zastosowanie praktyczne do leczenia zakaonych ran u pacjentw znajdujcych si na oddziaach szpi-talnych.

Sowa kluczowe: wycigi z Centella asiatica, aktywno antybiotyczna, przeciwutleniajca, muta-genna, dranica i przeciwzapalna

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