Ethnobotanical Leaflets 14: 654-64. 2010.

Antimicrobial Activities of Some Euphorbiaceae Plants Used in the Traditional Medicine of Akwa Ibom State of Nigeria

Uduak, A. Essiett¹, Kola’, K. Ajibesin*²

¹Department of Botany and Ecological studies, University of Uyo, Uyo, Akwa Ibom State, Nigeria

²Department of Pharmacognosy, Olabisi Onabanjo University, Sagamu Campus, Sagamu, Ogun State, Nigeria

Issued: June 1, 2010

Abstract

Nine plant species belonging to the Euphorbiaceae family and used in traditional medicine in Akwa Ibom State of Nigeria were evaluated for in vitro antimicrobial activity using agar diffusion method. The stem bark of Maesobotrya dusenii gave the most significant effect followed by its root bark. The inhibitory effect of M. dusenii stem bark extract (37 mm) on Pseudomonas aeruginosa was higher than that of Chloramphenicol (35 mm). However, Alchornea laxiflora leaf extract showed the weakest activity. The minimum inhibitory concentration of the extracts ranged between 12.5 and 250 µg/mL. The results of the antimicrobial effects validated the use of the plants to treat infections caused by these microorganisms.

Key words: Euphorbiaceae; antimicrobial activities; traditional medicine; Akwa Ibom State; Nigeria.

Introduction

The Euphorbiaceae is the 4^th largest family of the angiosperms comprising over 300 genera and about 7500 species distributed widely in tropical Africa (Gill, 1988). The euphorbiaceae plants are shrubs, trees, herbs or rarely lianas (Pandey, 2006). Many of them are xerophytes and cactoid and most often with milky latex. The family provides food (Pandey, 2006; Etukudo, 2003) and varied medicinal properties used in ethnobotany (Gill, 1988; Vasishta, 1974; Agbovie et al., 2002; Betti, 2004; Kubmarawa, 2007). They are useful in the treatment of ailments such as respiratory infections, venereal diseases, toothache, rheumatism, cough, ulcer and wounds (Oliver, 1960). However, some are also found as toxic. For instance, ricin contained in Ricinus communis is a well-known poisonous compound that elicits violent purgative action in man (Trease and Evans, 2002)), while the leaves of Euphorbia kamerunica are toxic to rats (Ajibesin, 2002). The plant is also a known irritant having in vitro cytotoxic activities (Abo and Evans, 1981).

In the traditional medicine of Akwa Ibom State, the under listed euphorbiaceae plants (Table 1) are used to treat various microbial diseases such as diarrhea, dysentery, skin infections and gonorrhea (Ajibesin et al., 2008). The antimicrobial uses of the plants in Akwa Ibom State, their ethnomedical uses in other parts of Nigeria, Africa and the world, as well as their chemical constituents are given.

Thus, this study aims at determining the antimicrobial effects of these euphorbiaceae plants, thereby validating their use in the traditional medicine of Akwa Ibom State, Nigeria.

Methods

Plant collection and authentication

Each fresh part (1 kg) of the plants (Table 1) was collected in 2006 and authenticated by Dr. U. Essiett of the Department of Botany and Ecological Studies, University of Uyo, Akwa Ibom State, Nigeria. Plant specimens were deposited in the herbarium of the Department of Pharmacognosy and Natural Medicine, University of Uyo, Uyo. The various plant parts were dried in the oven (30-65 ^oC), powdered by electric mill and stored.

Extraction

500 g of the respective plant part powder was macerated with 50% aqueous ethanol (2 x 5mL) for 72h at room temperature and filtered. The pooled liquid extract was concentrated to dryness in vacuo at 40^oC to give dry ethanol extract.

Phytochemical screening

The respective dry ethanol extracts were subjected to phytochemical screening using standard methods (Harborne, 1984; Sofowora, 1993) to show the classes of bioactive compounds in the plants.

Test organisms

The bacteria used in this study were typed cultures obtained from the Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria, while the fungi were clinical isolates collected from the same source. The bacteria: Staphylococcus aureus NCIB 8588, Bacillus subtilis NCIB 3610, Escherichia coli NCIB 86, Proteus vulgaris NCIB 67, Pseudomonas aeruginosa NCIB 950 and Klebsiella pneumoniae NCIB 418 were sustained on nutrient agar (Oxoid) slant at 4^oC prior to use. However, the fungi Candida albicans and Aspergilus flavus were sustained on Sabouraud’s Dextrose Agar (Oxoid) slants at 4^oC before use.

Antimicrobial susceptibility test

The dry ethanol extracts were evaluated against the test microorganisms using agar-gel diffusion method described by Alade and Irobi (1993). The ethanol extracts were redissolved in distilled water and tested at concentration level of 20 mg/ml. Fixed volumes (150μl) of the extracts and distilled water were separately introduced into equidistant wells (6 mm) bored on the surface of the agar and Sabouraud’s plates, which had been previously, inoculated with one of the test organisms. A well containing a standard drug, Chloramphenicol was made in the bacteria plates, while the fungal plates had a hole containing Nystatin as standard drug.

The bacteria were incubated at 37^oC for 24h, while the fungi were incubated at 25^oC for seven days. The presence of zones of inhibition surrounding the wells was taken as an evidence of antimicrobial activity.

Minimum inhibitory concentration

The minimum inhibitory concentration (MIC) was determined by incorporating various amounts (250 – 6.25 µg/mL) of the solution of extracts and fractions into sets of test tubes containing the culture media.50 µl of the standard test bacterial and fungal broth cultures were added into each of the test tubes. The set of tubes containing a mixture of bacteria and the sample (extracts and fractions) were incubated at 37 ^OC for 24 h, while those containing the fungi were incubated at 25 ^OC for 7 days.

A positive control tube containing only the growth medium of each of the organisms was also set up. The MIC was regarded as the lowest concentration of the extract or fraction that did not permit any visible growth when compared with that of the control tubes.

Results and Discussion

The different parts of the nine plants studied gave various classes of bioactive constituents (Table 2). All the plants contained cardiac glycosides, flavonoids, terpenes and saponins at varying concentrations, while tannins and phlobatannins were lacking only in M. dusenii root bark. Alkaloids were absent in M. fulvum leaves and root bark, M. barteri leaves, M. oppositifolius leaves and stem bark and M. dusenii root bark, while anthraquinones were absent in all the plants.

The extracts of the different parts of the plants gave varying degrees of antimicrobial effects (Table 3). The stem bark extract of M. dusenii elicited the most significant activity, while A. laxiflora leaf extract gave the least. Moreover, the M. dusenii stem bark extract showed the highest inhibitory effect against P. aeruginosa with a zone of inhibition of 37 mm. This inhibitory effect was higher than the Chloramphenicol effect against the same organism. Only the extracts of R. heudelotii stem bark, T. conophora leaves, A. laxiflora leaves and M. dusenii stem bark and root bark were active against A. flavus.

The result of this study showed that the different parts of all the plants elicited different levels of antimicrobial activities against all the test organisms. The extract of M. dusenii stem bark and root bark exhibited significant inhibitory effects against the entire test organisms (p < 0.01), while A. laxiflora leaf extract and M. fulvum leaf and root bark extracts gave weak activities. The other plants elicited moderate activities. However Ogundipe (2001) reported good antimicrobial activity for A. laxiflora leaf extract and identified quercetin, rutin and quercitrin as the flavonoids responsible for its activity. Similarly, the good antimicrobial effects recorded for E. heterophylla and R. heudelotii in this study are consistent with previous reports on the plants (Falodun et al., 2003; Momeni et al., 2005).

The results of the antimicrobial effects of all extracts showed that some of the plant species might be weak antimicrobial plants, while others may be potent. Similar observation has been reported for the antimicrobial activities of some Euphorbiaceae plants (de Lima et al., 2006; Parekh and Chanda, 2007). Also, antimicrobial screening of some plants with ethnobotanical antimicrobial uses was reported to show similar result (Rajakaruna et al., 2002).

Minimum inhibitory concentration values of 12.5 - 250 µg/mL were recorded for the extracts of some of the Euphorbiaceae plants against the test bacteria, while a range of 25 – 250 µg /mL was obtained against Candida albicans and Aspergillus flavus (Table 4). The result of minimum inhibitory concentration of extracts showing 12.5 and 25 µg /mL suggests that the extracts may act as bactericidal and fungicidal agents to these microorganisms.

The bioactive compounds responsible for the inhibitory effects of these plants were detected in their phytochemical screening, some of which were reported in literature as antimicrobial constituents. Flavonoids are known to be antimicrobial in nature (Joseph et al., 2002). Flavonoids isolated from the leaves of Euclea crispa sub sp. crispa were reported to give antimicrobial activity (Pretorius et al., 2003). Tannins identified from Vaccinium vitis-idaea, terpenes from Vernonia amygdalina and saponins from Allium minutifolium and were all established as antimicrobial constituents of the plants (Ho et al., 2001; Barile et al., 2007).

The antimicrobial effects of the plants, established in the results, have lent credence to the ethnobotanical use of these euphorbiaceae plants in treating infections caused by these organisms.

In conclusion, this study showed the antimicrobial activity of the extracts of various plants of the euphorbiaceae family used in ethnomedicine of Akwa Ibom State of Nigeria. The inhibitory effect of the plant extracts validated the medicinal use of the plants. Further work is required for identifying the active constituents of the plants.

Acknowledgments

The authors acknowledge the contributions of Mr. Patrick Iberi, a Technologist in the Department of Pharmacognosy and Natural Medicine, University of Uyo, Nigeria, and Mr. Etefia Okon, a Laboratory staff in the same Department.

References

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Table 1. Traditional uses of some Euphorbiaceae plants.

Plant species Source Traditional uses Chemical constituents Plant part

Used

Euphorbia Ab Purgative, malaria, rashes, Alkaloids, phenolics, saponins, Leaves

heterophylla L. respiratory tract infection (26). (27), resins, triterpenes (28).

Mallotus Es Lumbago (29), dysentery, wounds, Rottelerin, flavonols (28, 34). Leaves,

Oppositifolius measles, whitlow (5), worms (30), stem bark.

(Geisel.) Mull. Arg. laxative, vermifuge, aphrodisiac

headache (31, 28, 32).

Macaranga barteri In Ulcer, stomatitis (34), amnesia (35). Phenolics (36) Leaves

Mull. Arg

Maesobotrya dusenii Ek Diarrhoea, dysentery(3). Stem bark,

(Pax) Hutch. root bark.

Phyllanthus amarus Ik Gall stone, kidney stone (37), boil, Phyllanthin, hypophyllanthin, Whole

Schum. & Thonn. sores, skin disease, pile (30). (38), inulin, saponins (39), plant

flavones (28).

Ricinodendron Ur Constipation, miscarriage, Aleuritolic acid, phenolic (17). Stem bark

heudelotii painful menstruation,

(Baill.) Pierre ex Pax (32), female infertility (5),

diarrhoea, dysentery (40),

asthma (41).

Tetracarpidium It Hiccups (42), fibroid, female Leaves

conophorum infertility, irregular menstrual

(Mull. Arg.) Hutch Flow (30), skin rash, dysentery

& Dalz.

Alchornea laxiflora Uy Oral hygiene, malaria (42), Terpenoids (44),

(Benth.) Pax K. infectious disease, quercetin, rutin,

Hoffm. inflammation (43,44). quercitrin (38). Leaves

Manniophyton Or Insanity (35), skin

infection Terpenoids (45) Leaves,

fulvum root bark.

Mull. Arg.

Ab = Abak, Es = Eastern Obolo, In = Ini, Ek = Eket, Ik = Ikono, Ur = Uruan, It = Itu, Uy = Uyo, Or = Oron, 26 = Falodun et al., 2006; 27 = Falodun and Agbakwuru, 2007; 28 = Iwu, 1986; 29 = Abiww, 1990; 30 = NNMDA, 2006 a; 31 = Lewis and Elvin-Lewis, 1977; 32 = Burkill, 1994; 34 = Adjanohoun et al., 1981; 35 = Bouquet, 1969; 36 = Adesegun et al., 2007; 37 = Anonymous, 2003; 38 = Sharma et al., 1993; 39 = Gill, 1992; 40 = Cunningham, 1993; 41 = Barnish and Samai, 1992; 42 = Walker and Sillans, 1995; 43 = NNMDA, 2006 b; 44 = Farombi et al., 2003; 45 = Nia et al., 2005; 3 = Etukudo Etukudo, 2003; 5 = Agbovie et al., 2002; 17 = Momeni et al., 2005.

Table 2 Phytochemical screening of some Euphorbiaceae plants.

Metabolites

E.h

M.o

M.d

M.b

P.a

R.h

T.c

A.l

M.f

Saponins

Tannins

Flavonoids

Alkaloids

Phlobatannins

Anthraquinones

(Borntrager’s test)

Cardiac glycosides

(i) Lieberman

(ii) Salkowski

(iii) Keller Kiliani

Terpenes

+++

L Sb

+++ ++

++ ++

- -

+++ ++

- -

++ ++

++ +++

+++ ++

++ +++

Sb Rb

++ ++

- -

++ +++

- -

+ ++

++ ++

++ +++

++ ++

+++

Rb L

++ +

+++ +++

++ +++

- -

+++ ++

- -

++ +++

+++ ++

++ +++

E.h = Euphorbia heterophylla; M.o = Mallotus oppositifolius; M.b = Maesobotrya dusenii; P.a = Phyllanthus amarus; R.h = Ricinodendron heudelotii; T.c = Tetracarpidium conophora; A.l = Alchornea laxiflora; M.f = Maniophyton fulvum.

L = Leaves; Sb = Stem bark; Rb = Root bark; Wp = Whole plant.

Table 3 Antimicrobial effects of the extracts of some Euphorbiaceae plants.

Plant Species Plant part Microorganisms (Inhibition zone in mm)^a

E. heterophylla

M. oppositifolius

M. dusenii

M. barteri

P. amarus

R. heudelotii

T. conophora

A. laxiflora

M. fulvum

Chloramphenicol (4

μg/mL)

Nystatin (4 μg/mL)

Leaves

Stem bark

Root bark

Leaves

Whole plant

Stem bark

Leaves

Root bark

B.s

21±1.4*

31±1.7*

25±3.6*

25±1.0*

30±1.7*

14±1.4

2±0.0

35±0.0*

E.c

15±1.7

23±3.6*

16±1.2

17±1.0

21±1.2*

13±0.0

18±1.6*

6±0.0

20±2.0*

3±1.0

28±0.0*

S.a

28±2.0*

33±1.0*

14±0.0

23±1.7*

24±2.0*

7±0.0

5±1.0

9±0.0

11±2.0

15±1.7

37±0.0*

P.a

10±1.0

31±2.0*

22±2.4

37±1.0*

23±1.4*

25±1.6*

15±1.9

6±2.0

15±3.6

8±2.4

35±0.0*

K.p

25±2.6*

30±2.0*

21±2.0

20±2.4*

26±2.6*

21±1.7*

32±0.0*

P.v

30±2.0*

24±1.4*

16±0.0

13±0.0

17±1.2

28±2.0*

10±1.0

10±1.4

20±2.4*

11±0.0

6±2.0

13±1.2

33±0.0*

C.a

20±1.4*

28±2.0*

25±1.7*

31±2.0*

23±2.0*

30±1.2*

9±0.0

10±1.2

4±1.0

16±2.6*

16±2.0*

28±0.0*

A.f

8±0.0*

7±1.0

25±1.0*

9±1.2

9±0.0

34±0.0*

B.s = B. subtilis NCIB 3610; E.c = E. coli NCIB 86; S.a = S. aureus NCIB 8588; P.a = Ps. aeruginosa NCIB 950; K.p = K. pneumoniae NCIB 418; P.v = P. vulgaris NCIB 67; C.a = C. albicans; A.f = A. flavus.

a = Values are the mean of quadruplicate readings; P<0.01.

Saline solution (150 μL) = - ; - = No inhibition zone

Table 4 Minimum inhibitory concentration of the extracts of some Euphorbiaceae plants.

Plant Species Plant part Microorganisms (Inhibition zone in mm)^a

E. heterophylla

M. oppositifolius

M. dusenii

M. barteri

P. amarus

R. heudelotii

T. conophora

A. laxiflora

M. fulvum

Chloramphenicol (4

μg/mL)

Nystatin (4 μg/mL)

Leaves

Stem bark

Root bark

Leaves

Whole plant

Stem bark

Leaves

Root bark

B.s

250

>250

12.5

E.c

200

250

100

>250

200

>250

100

>250

12.5

S.a

12.5

>250

100

>250

250

6.25

P.a

250

100

12.5

200

>250

250

>250

12.5

K.p

100

P.v

250

>250

250

>250

100

>250

12.5

C.a

100

>250

250

12.5

A.f

>250

250

6.25

a = Values are the mean of quadruplicate readings

- = No inhibition