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
1

Research Institute of Medicinal Plants,

ul. Libelta 27, 61-707 Pozna, Poland
2
Department of Pharmacology, University of Medical Sciences,
ul. Rokietnicka 5a, 60-806 Pozna, Poland
3
Department 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: jwojcik@iripz.pl
Summary
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 carrageen after 3 h from application (ca. 12%).
We conclude that the topically applied preparation (cream or gel) containing 5% of investigated 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 antinflammatory activity.

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


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 extracts 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 asiatica (L.) Urban appear to have many valuable biological properties, especially wound 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 asiaticoside, phenolic compounds with flavonoids and volatile oil. Moreover, the antitumor, 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 Cicatridine (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 Centella 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 obtained with 30% ethanol (C2 and C5) and aqueous-alcoholic extracts obtained with
60% ethanol (C3 and C6).

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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 including 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 performed with tetracycline (Merck).

Antioxidant properties
The studies were performed in vitro. The test with DPPH (2,2-diphenyl-1-picrylhydrazyl) 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 Grampositive 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 Mueller-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

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


extract. The plates were incubated at the temperature of 370C and after 24 h the
minimal inhibitory concentration (MIC) of extract inhibiting development of investigated 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 ammonium 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, protected 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).
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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 suspension 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 aqueous 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 concentration 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 (Carrageenan 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 investigated 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 investigated 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. (immediately 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),

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


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-alcohol extracts obtained with 30% ethanol) inhibited the development of standard
strain Staphylococcus aureus FDA 209 P in concentration of 1000 g/ml. Other investigated 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
C1
C2
C3
C4
C5
C6
tetracycline

MIC (g/ml)
>1000
1000
>1000
>1000
1000
>1000
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 stronger antioxidative activity in comparison to other extracts. For example, chlorogenic acid was active in concentration of 0.01 g/ml.
Ta b l e 2 .
Antioxidative activity of herb Centella asiatica extracts.
investigated extracts
C1
C2
C3
C4
C5
C6
chlorogenic acid

MIC (g/ml)
10
10
1
10
10
1
0.01

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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 obtained results indicate that Gram-positive bacteria are inhibited by a little higher
concentration of extract from Centella asiatica (MIC mean=30 mg/ml) in comparison to Gram-negative bacteria (MIC mean=23 mg/ml). The highest resistance
(MIC 2540 mg/ml) showed the strains of Staphylococcus aureus and Corynebacterium 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 characterized 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 Gramnegative bacteria isolated from wounds.
strain
Gram-positive bacteria
1. Staphylococcus aureus
2. Staphylococcus aureus
3. Staphylococcus aureus
4. Staphylococcus aureus
5. Staphylococcus aureus
6. Staphylococcus aureus
7. Staphylococcus aureus
8. Staphylococcus epidermidis
9. Streptococcus pyogenes
10. Enterococcus faecalis
11. Enterococcus faecalis
12. Enterococcus faecalis
13. Corynebacterium jejkeum
Gram-negative bacteria
14. Escherichia coli
15. Escherichia coli
16. Escherichia coli
17. Escherichia coli
18. Escherichia coli
19. Escherichia coli
20. Klebsiella pneumoniae
21. Pseudomonas aeruginosa

place of isolation

MIC(mg/ml)

decubitus
wound in anal area
frontal cranium absces
wound on ear concha
decubitus
perirectal fistula
burn wound
acne
perirectal fistula
decubitus
wound
purulent by catheter outlet
wound after cardiosugery

30.0
35.0
35.0
25.0
35.0
40.0
35.0
15.0
10.0
25.0
20.0
10.0
35.0
25.0
20.0
25.0
25.0
25.0
25.0
25.0
15.0

wound after cardiosugery

decubitus
wound after cardiosugery
perirectal fistula
perirectal fistula
purulent by catheter outlet
wound after cardiosugery
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.

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


Ta b l e 4 .
Studies on mutagenic activity of aqueous-ethanol extract from Centella asistica (C1).
substrates added to
surface medium B

name of plate

number of colonies of
S.typhimurium on the plate

I, IV
<100*
1. growth control of S. typhimurium
2. growth control of S. typhimurium in presence of mutagen (natrium azide)
I, II.IV
>1000
3. investigated extract from C. asiatica (10 mg/plate)
I, III.IV
<100*
4. investigated extract from C. asiatica (1 mg/plate)
I, III.IV
<100*
I, III.IV
<100*
5. investigated extract from C. asiatica (0.1 mg/plate)
* spontaneous mutations
I mixture of histidine and biotine, II solution of natrium azide, III solution of investigated extract, IV dilution of
S. typhimurium cultivation.
Explanations: see Methods.

The estimation of irritative activity shows that topical application of extract

from Centella asiatica as well of 1% cream as of 1% aqueous suspension in dosage
of 5 mg does not induct oedema, erythema or scabs.
Other results are presented in Table 5. It could be stated that the aqueous
extract from Centella asiatica after the topical application in used dosage exhibits a poor antiinflammatory activity after a longer time (about 12% of oedema
reduction after 3 h). The antiinflammatory activity could be not compared with
the efficacy of indometacine a representative of nonsteroidal antiinflammatory
remedies. It could be supposed that the antiinflammatory activity of investigated
extract could be higher after the increase of concentration to 5% in preparation.
The further pharmacological studies should be continued.
Ta b l e 5 .
Effect of aqueous-ethanol extract from Centella asiatica in form of 1% creme on rat paw oedema.
time of activity of 1% creme with
aqueous-ethanol extract from C.
asiatica (h)
1.5
3.0
n number of animals in group

rat paw oedema

control
(n=7)

indometacine (10 mg/kg

body weight, n=8)

extract from C. asiatica (5

mg/kg body weight, n=7)

0.260.06
0.460.06

0.180.05
0.120.04

0.270.04
0.360.07

CONCLUSIONS
1. The aqueous-ethanol extracts (30% ethanol) from Centella asiatica (C2 and C5)
have a stronger antibiotic activity as compared to others extracts. In comparison with tetracycline the antibiotic activity of above extracts can be determined as a poor one.
2. The aqueous-ethanol extracts (60% ethanol) showed a stronger antioxidative
activity compared to other extracts. In comparison with chlorogenic acid the
antioxidative activity of above extracts is poor.
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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) containing 5% of investigated aqueous-ethanol extract from herb of Centella asiatica (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.

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11. Centella asiatica selected triterpenes. Indena. http://www/indena.it.
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14. De Sanctis MT, Belcaro G, Incandela L, Cesarone MR, Griffin M, Ippolito E, Cacchio M. Treatment of
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Centella asiatica: a clinical, prospective, placebo-controlled, randomized, dose-ranging trial. Angiology
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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
1
Instytut Rolin i Przetworw Zielarskich,
Libelta 27,
61-707 Pozna
2
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: jwojcik@iripz.pl

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Streszczenie
Wycigi wodno-alkoholowe (30% etanolu) z ziela Centella asiatica odznaczay si silniejszym 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 bakteriostatyczne 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% zawiesiny 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 wodnoalkoholowego z ziela Centella asiatica (50 mg/g preparatu), moe znale zastosowanie
praktyczne do leczenia zakaonych ran u pacjentw znajdujcych si na oddziaach szpitalnych.
Sowa kluczowe: wycigi z Centella asiatica, aktywno antybiotyczna, przeciwutleniajca, mutagenna, dranica i przeciwzapalna