Anti-inflammatory effects of crude methanolic extract and fractions of Alchornea cordifolia leaves

Journal of Ethnopharmacology 89 (2003) 19–24

Anti-inflammatory effects of crude methanolic extract and fractions of Alchornea cordifolia leaves P.O. Osadebe∗ , F.B.C. Okoye Department of Pharmaceutical Chemistry, Faculty of Pharm. Sciences, University of Nigeria, Nsukka, Nigeria Received 3 September 2002; received in revised form 4 April 2003; accepted 7 May 2003

Abstract The leaves of Alchornea cordifolia were collected, identified, dried, and reduced to coarse powder and extracted with aqueous methanol. Using various solvent treatments, the powdered dried leaf was fractionated into five fractions, A1, A2, B, C, D and E. The fractions were subjected to phytochemical analysis to identify the biologically active constituents. The anti-inflammatory effects of crude methanolic extract (ME) of Alchornea cordifolia leaves and the five fractions were evaluated using egg-albumen-induced rat hind paw oedema as a model of inflammation. The crude extract was also subjected to acute toxicity test. Fraction A2, which exhibited the most promising anti-inflammatory effect, was also subjected to analgesic and ulcerogenic tests. Phytochemical analysis of the extracts showed the presence of terpenes, sterols, flavonoids, tannins, carbohydrates, glycosides, saponins and traces of alkaloids. The LD50 of the aqueous ME was found to be 1131.4 mg/kg. The crude ME (50 mg/kg) gave anti-inflammatory activity which was significant (P < 0.05) at all the observation times (1–3 h). The different solvent fractions exhibited varying degrees of anti-inflammatory activities, with terpenoid fraction (A2) and the tannin-containing multi-component fraction (D) showing very high and significant (P < 0.01) activity at 100 mg/kg, with percentage inhibition of oedema value of 87.69 each. In conclusion, the aqueous ME of Alchornea cordifolia leaves could be beneficial in the management of different inflammatory disease states. Its anti-inflammatory activity may not be attributed only to the terpenoid content. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Alchornea cordifolia; Anti-inflammatory; Analgesic; Aqueous methanolic extract; In egg-albumen-induced oedema

1. Introduction Generally, the inflammatory process involves a series of events that can be elicited by numerous stimuli such as infectious agents, ischaemia, antigen–antibody interaction and thermal or physical injury (Insel, 1990). It is usually associated with pain as a secondary process resulting from the release of algesic mediators (Hunskaar and Hole, 1987). Therapy of inflammatory diseases is usually directed at the inflammatory processes. Through years of ingenious syntheses and structural modifications, which usually accompany design and development of new drug substances, many non-steroidal anti-inflammatory agents (NSAIDS) have been prepared and marketed (Olaniyi, 2000). These have been of immense help in the management of various inflammatory conditions like rheumatism, arthritis and breast pain. However, these drugs are known to provoke gastrointestinal irritation. This makes them widely unacceptable, especially in the elderly where the disease is more prevalent, hence the

∗ Corresponding

author.

search for alternative anti-inflammatory drugs and medicines among the bounties of natural herbs. Moreover, in the face of rising cost of orthodox medicines, phytomedicinal treatment of diseases has become the order of the day in most parts of Africa, chiefly because of their ready affordability and availability, especially in the rural set-ups where the greater percentage of the people are poor and merely subsisting. Alchornea cordifolia, Schum-Thron (family, Euphorbiaceae) is a shrub found along the coastal regions of West Africa. The plant is an important crude drug in the indigenous system of medicine for the management of pain, rheumatism, and arthritis, pile, toothache and some other inflammatory disease states. In all the conditions, the ground leaves is applied to the aching places and wounds. Crushed leaves of Alchornea castaneifilia are rubbed on painful joints as an analgesic and beaten into a paste and applied to painful wounds. The leave decoction of Alchornea cordifilia is used as an eye lotion and as an antibacterial in the treatment of common cold, cough and diarrhoea (Van Medendach de Roy, 1996). A decoction of the leafy twigs is applied as a wash for feverish chills, rheumatic pains and sores, and as an application for sore feet as lotion.

0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00195-8

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P.O. Osadebe, F.B.C. Okoye / Journal of Ethnopharmacology 89 (2003) 19–24

The young leaves are applied with pepper and white clay as an enema to prevent abortion (Bennett, 1950). The powdered leaves are mixed with palm wine for the treatment of gonorrhoea. The root and stem bark are used in the treatment of Jaundice. The dry leaves are used to prepare hypoglycaemic infusions that are drunk after meals. The leaves are used internally for the management of gastrointestinal, respiratory and urinary tract infections and externally for wounds (Iwu, 1983). An extract of the roots of Alchornea floribunda prepared by macerating the plant material for several days in palm wine is used in Zaire as a stimulating intoxicant and aphrodisiac. Currently, in Peruvian herbal medicine, Alchornea castaneifilia (Iporuru) is widely used to treat impotency and for reducing sugar in the blood and urine in the diabetics. For this purpose, one cup of dried leaves is infused in 500 ml of water for 1 day and two to three doses (of one-half cup per dose is drunken daily for impotency). For diabetes, one-half of cup of dried leaves is infused in 1 litre of water and one cup is drunk at each meal. Some constituents of the plant, Alchornea cordifolia, have been identified, mainly tannins, alkaloids and epoxyacids (Bennett, 1950; Kleiman et al., 1997). The pharmacological activities already investigated include anti-microbial activity, anti-amoebic, antidiarrhoeal and spasmolytic effects (Ebi, 2001; Tona et al., 2000). Hitherto, there is no scientific study confirming the anti-inflammatory activity. The present study is aimed at establishing anti-inflammatory activity, and the nature or chemical classes of the constituents responsible for the anti-inflammatory activity. This will serve as the basis for further detailed chemical and pharmacological investigation of the active principles. Moreover, it is known that pain secondary to an inflammatory process is one of the cardinal manifestations of this inflammatory disorder (Santos et al., 1996). Hence, its evaluation in an anti-inflammatory agent is necessary.

2. Materials and methods 2.1. Plant materials Green leaves of Alchornea cordifolia, Schum-Thron (Family, Euphorbiaceae) were collected during the month of August 2001, at Okpi, Nsukka, Enugu State, Nigeria. Mr. Ozioko of Bioresource Development and Conservation Project of the same University authenticated the materials. A voucher specimen was deposited at the Herbarium (Herbarium No. = 012/01) of the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences of same University. 2.2. Chemicals n-Hexane, chloroform, ethylacetate, methanol, absolute ethanol, and Tween 20. All the solvents were BDH grade and were distilled before use.

2.3. Animals Wister rats (120–180 g) and albino mice (22–25 g) were purchased from Faculty of Veterinary Medicine, University of Nigeria. The animals were stored in standard laboratory conditions and fed with rodent commercial diet (Guinea Feed Nig. Ltd.) and water ad libitum. Food was withdrawn 6 h before the tests. 2.4. Extraction A preliminary extraction of the powdered dried leaves was performed by macerating 20 g of the plant material with 100 ml of aqueous methanol (70%) for 24 h at room temperature (22 ◦ C). This methanolic extract (ME) was used for the toxicity test and the preliminary anti-inflammatory study. The powdered leaves (600 g) were extracted with 3.5 l of distilled n-hexane. The resulting extract, A, was concentrated in vacuo. The air-dried residue of A was repeatedly extracted with 90% methanol. The methanol extract (90%) was treated (washed) in succession with chloroform and ethylacetate to yield chloroform soluble (B), ethylacetate soluble (C) and ethylacetate insoluble (D). Another n-hexane extract, obtained from the extraction of a second 600 mg portion of powdered leaves with 3.5 l of distilled n-hexane, was further separated into fractions, A1 and A2 by precipitating the concentrated n-hexane solution with absolute ethanol to obtain a new hexane residue (A1) and ethanol fraction (A2). All the fractions (except A1, which was too difficult to solubilise) were solubilised with 8% Tween 20 and tested for anti-inflammatory activity. The yield of the extractions and fractions are shown in Table 1. 2.5. Phytochemical tests Phytochemical tests were carried out on the aqueous ME and on all the solvent extracts using the procedure outlined by Harbourne (1984). In general, tests for the presence or absence of phytochemical compounds using the above methods involve the addition of an appropriate chemical agent to both the crude material and the ethanolic extracts of the plant in a test tube. The mixture is then shaken vigorously Table 1 Result of extraction of the plant material using different solvents Solvent fraction

Yield

Chemical constituents

w/w

Percent (%)

A1 A2 B C D

1.41 5.26 6.21 3.19 51.87

0.24 0.88 1.04 0.53 8.66

Resins, triterpenoids (traces) Triterpenoids Triterpenoids, flavonoids (traces) Flavonoids, phenolics, glycosides Tannins, saponins, carbohydrates, glycosides, alkaloids (traces)

Chloroform soluble (B), ethylacetate soluble (C) and ethylacetate insoluble (D). A1 and A2 are fractions obtained by precipitating n-hexane extract (A), with absolute ethanol.

P.O. Osadebe, F.B.C. Okoye / Journal of Ethnopharmacology 89 (2003) 19–24

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or gently as the case may be. The presence or absence of saponins, flavonoids, tannins, alkaloids, etc. was observed.

ulceration. Aspirin (200 mg/kg) and 5% Tween 20 were used as positive and negative controls, respectively.

2.6. Anti-inflammatory test

2.9. Acute toxicity test (LD50 determination)

Egg-albumen-induced rat paw oedema was used in the study (Ekpendu et al., 1994). The paw oedema was induced in the plantar region of the rat right hind paw. The animals (n = 5) were fasted for 6 h and deprived of water only during the experiment. The deprivation of water was to ensure uniform hydration and to minimize variability in oedematous response (Winter et al., 1963). They were given intraperitoneal injection of the extracts and the fractions solubilised in 8% Tween 20 at doses of 50, 100 and 200 mg/kg. The control groups were treated with 0.2 ml of Tween 20 (negative control), 100 mg/kg aspirin (positive control) and 10 mg/kg hydrocortisone (positive control). All the substances were administered intraperitoneally 30 min before the intradermal injection of the phlogistic agent (0.1 ml of fresh undiluted egg albumen) in the plantar region of the right hind paw. Paw volume was measured by water displacement method using a Plethysmometer (Ugo Basil) at 0, 1, 2, 3 and 4 h after egg-albumen injection. The anti-inflammatory activity was calculated at each time of observation as percent inhibition of oedema in the animals treated with substances under test, in comparison to the vehicle-treated animals. The percentage inhibition of oedema is calculated using the formula,

The acute toxicity test (LD50 ) of the crude ME was determined according to the procedure described by Lorke 1983). The crude aqueous ME was used for the test. Albino mice (20–25 g) of either test were used. This method involved an initial dose finding procedure, in which the animals were divided into three groups of three animals per group. Doses of 10, 100 and 1000 mg/kg were administered intraperitoneally (i.p.), one dose for each group. The treated animals were monitored for 24 h for mortality and general behaviour. From the result of the above step, four different doses of 200, 400, 800 and 1600 mg/kg were chosen and administered (i.p.) respectively to four groups of one mouse per group. The treated animals were again monitored for 24 h. The LD50 was then calculated as the geometric mean of the lowest dose showing death and the highest dose showing no death.

% Inhibition =

V0 − Vt × 100 V0

where Vt is the volume of oedema at corresponding time, and V0 is the volume of oedema of control rats at the same time. 2.7. Assessment of analgesic activity of fraction A2 Acetic acid-induced writhings in mice was used for the study. A 0.1 ml portion of acetic acid (1%) was injected intraperitoneally to elicit a writhing syndrome in mice (n = 5). Fraction A2 (100 mg/kg) was administered intraperitoneally 30 min before acetic acid and the number of writhes displayed from 5 to 30 min after acetic acid injection were counted (Williamson et al., 1996). In this experiment, acetyl salicylic acid (ASA, 100 mg/kg) served as the reference drug. 2.8. Assessment of ulcerogenic activity of fraction A2 The test was carried out according to Cashin et al. (1979). Fraction A2 (400 mg/kg) was administered orally to rats after 24-h fasting. The animals were killed 3 h after dosing, and the stomach was removed and cut along the lesser curvature. The mucosa surface was washed with normal saline and observed with magnifying lens to determine the degree of

3. Statistical analysis Data were analysed using Student’s t-test (Woodson, 1987). 4. Results 4.1. Extraction and fractionation The preliminary extraction of Alchornea cordifolia leaves (20 g) with aqueous methanol afforded extract, which on phytochemical analysis, showed the presence of terpenes, sterols, flavonoids, tannins, carbohydrates, glycosides, saponins and traces of alkaloids. Extraction of the plant material using different solvents gave five fractions with varying phytochemical constituents. The fractions, the yields, and their different phytochemical constituents are shown in Table 1. 4.2. Anti-inflammatory tests The result of anti-inflammatory evaluation of extracts of Alchornea cordifolia is shown in Tables 2 and 3. The aqueous ME of Alchornea cordifolia leaves administered at a dose of 50 mg/kg, induced a significant (P < 0.05) oedema inhibition (68.25%) at 3 h, whereas ASA (100 mg/kg) and hydrocortisone (10 mg/kg) induced 69.23 and 72.30% inhibition of oedema, respectively. Fractions A2, B and D showed significant anti-inflammatory activity at 100 mg/kg. Fraction C showed significant (P < 0.02) activity at this dose level only at 3 h after treatment. At 50 mg/kg, only fraction A2 showed significant (P < 0.05) anti-inflammatory

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Table 2 Effect of the ME and solvent fractions of n-hexane and 90% MEs of Alchornea cordifolia on egg-white-induced paw oedema in rats Extract/fraction

Dose

Oedema volume in ml (mean ± S.E.M.)

MEa A2 A2 A2 B B C C D D ASA Hydrocortisone 8% Tween 20

50 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg 50 mg/kg 100 mg/kg 50 mg/kg 100 mg/kg 50 mg/kg 100 mg/kg 100 mg/kg 10 mg/kg 0.2 ml

0.63 0.50 0.25 0.20 0.75 0.40 0.72 0.55 0.65 0.30 0.30 0.33 0.75

1h

2h ± ± ± ± ± ± ± ± ± ± ± ± ±

0.27∗ 0.03 0.05∗∗ 0.01∗∗∗ 0.01 0.03 0.15 0.07 0.10 0∗ 0.04∗ 0.10 0.16

3h

0.25 0.48 0.20 0.08 0.58 0.20 0.70 0.52 0.60 0.20 0.30 0.23 0.65

± ± ± ± ± ± ± ± ± ± ± ± ±

0.02∗∗∗∗∗ 0.04 0.05∗∗∗ 0.01∗∗∗∗∗ 0.01 0.02∗∗∗ 0.13 0.02 0.10 0.02∗∗∗ 0.04∗∗ 0.07∗∗ 0.10

0.20 0.37 0.37 0.08 0.48 0.15 0.49 0.30 0.56 0.08 0.20 0.18 0.65

± 0.02∗∗∗∗∗ ± 0.03∗ ± 0.03∗ ± 0.01∗∗∗∗∗ ± 0.01 ± 0∗∗∗∗ ± 0.04 ± 0.05∗∗ ± 0.07 ± 0.01∗∗∗∗∗ ± 0∗∗∗∗ + 0.07∗∗∗∗ ± 0.10

Chloroform soluble (B), ethylacetate soluble (C) and ethylacetate insoluble (D). A1 and A2 fractions were obtained by precipitating the concentrated n-hexane solution with absolute ethanol. a For the ME, test of significance was carried out against negative control (5% Tween 20) which gave oedema volumes of 1.00 ml at 1 h, 1.00 ml at 2 h and 0.63 ml at 3 h after treatment. ∗ Significant at P < 0.05. ∗∗ Significant at P < 0.02. ∗∗∗ Significant at P < 0.01. ∗∗∗∗ Significant at P < 0.002. ∗∗∗∗∗ Significant at P < 0.0001.

activity. Fraction A2 was found to show a dose-dependent anti-inflammatory effect with ID50 (dose giving 50% inhibition) of 79.4 mg/kg at 2 h. 4.3. Analgesic effect of A2 Fraction A2, at a dose of 100 mg/kg also exhibited significant (P < 0.05) analgesic activity comparable to the reference drug ASA (Table 4). Table 3 Percentage inhibition of oedema by the solvent fractions and 90% MEs of Alchornea cordifolia Extract/fraction

ME A2 A2 A2 B B C C D D ASA Hydrocortisone 8% Tween 20

Dose

50 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg 50 mg/kg 100 mg/kg 50 mg/kg 100 mg/kg 50 mg/kg 100 mg/kg 100 mg/kg 10 mg/kg 0.2 ml

Percentage oedema inhibition at corresponding times 1h

2h

3h

37a

75a

33.3 66.67 73.33 0 46.67 4.00 26.67 13.33 60.0 60.00 56.00 –

26.15 69.33 80.00 10.77 69.23 10.06 26.67 7.69 69.23 53.85 64.60 –

68.25a 43.08 87.69 87.09 26.15 76.92 24.6 20.00 13.85 87.69 69.23 72.30 –

Chloroform soluble (B), ethylacetate soluble (C) and ethylacetate insoluble (D). A1 and A2 fractions were obtained by precipitating the concentrated n-hexane solution with absolute ethanol. a Percentage inhibition here was calculated against oedema volume of 5% Tween 20 (1.00 ml) at corresponding time.

Table 4 Analgesic activity of fraction A2 in mice Fraction

Dose

No. of writhes (mean ± S.E.M.)

Percentage analgesia

A2 ASA Vehicle

100 mg/kg 100 mg/kg 0.1 ml

7.50 ± 0.45 12.35 ± 0.84 24.40 ± 0.75

69.26 49.39 –

Percentage analgesia =

No. of writhes (vehicle)−No. of writhes (A2) ×100. No. of writhes (vehicle)

4.4. Ulcerogenic effect Fraction A2 at 400 mg/kg did not show marked gastric irritation compared to ASA (200 mg/kg, p.o.). The ulcer index was 0.05 ± 0.01 as against that ASA (1.5 ± 0.40). 4.5. Acute toxicity test (LD50 ) The result of the acute toxicity studies is shown in Table 5. The LD50 of the aqueous ME was calculated to be 1131.4 mg/kg.

5. Discussion The intraperitoneal LD50 value of 1131.4 mg/kg, obtained for the aqueous ME falls within the practically non-toxic range (Loomis, 1978). Doses up to 800 mg/kg were found to be practically safe, resulting in no deaths of the mice (Table 5). The acute toxicity test method by Lorke (1983) utilises less number of mice (usually 13), and provides a 24-h LD50 figure, which is adequate for most practical purposes.

P.O. Osadebe, F.B.C. Okoye / Journal of Ethnopharmacology 89 (2003) 19–24 Table 5 Acute toxicity test of the crude ME of Alchornea cordifolia Dose (mg/kg)

Death pattern at 24 h after treatment

Stage I 10 100 1000

0/3 0/3 0/3

Stage II 200 400 800 1600

0/1 0/1 0/1 1/1

LD50 = (800 × 16, 000)1/2 = 1131.4 mg/kg.

Based on the above result, doses of 50, 100 and 200 mg/kg were selected for the anti-inflammatory screening. The marked anti-inflammatory activity (P < 0.001) exhibited by the aqueous ME and its observed low toxicity validates its use in indigenous medicine for the management of pile, rheumatism, arthritis, pains, etc. The different solvent fractions exhibited varying degrees of anti-inflammatory activities, with terpenoid fraction (A2) and the polyphenolic fraction (D) showing very high and significant (P < 0.01 and P < 0.001, respectively) activity at 100 mg/kg. Fraction B, which contains traces of terpenoids and flavonoids also exhibited significant (P < 0.05) activity at this dose level, while fraction C, the flavonoids fraction exhibited anti-inflammatory activity at the same dose level, which was only significant at 3 h after treatment. This latter observation is not consistent with the popular view that flavonoids are mainly responsible for the anti-inflammatory activity exhibited by plant extracts (Gabor, 1973). Only fraction A2 showed significant (P < 0.05) anti-inflammatory effect at 50 mg/kg. At 2 h, its anti-inflammatory effect was found to be dose-dependent, with an ID50 value of 79.4 mg/kg. It also showed a good analgesic effect at a dose of 100 mg/kg with little or no gastrolessive effect at 400 mg/kg (p.o.). The analgesic activity exhibited by fraction A2 suggests an added advantage in the use of the extract as an anti-inflammatory agent, since most inflammatory conditions are usually associated with pain as a secondary process resulting from the release of algesic mediators (Hunskaar and Hole, 1987). The observation that the most active fraction showed virtually no gastrolessive effect even at 400 mg/kg precludes the inhibition of prostaglandin synthesis as a possible mechanism of action (Barar, 2000; Vane and Botting, 1990). Though this assertion is rarely conclusive, the observed effectiveness of the agent in the acute exudative phase of inflammation suggests the inhibition of histamine and/or serotonin as a likely mechanism of action (Willoughby and Fllower, 1993). It has been reported that egg-albumeninduced inflammation is similar to that produced by carrageenin (Williams and Morley, 1973; Akah et al., 1993). However, in the present investigation, it was observed that inflammation induced by egg albumen was not sustained

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beyond 4 h. This may be explained in part by the absence of sulphated sugars in egg albumen, which are found in carrageenin, and are responsible for activation of complement systems and other latter phase inflammatory mediators (Williamson et al., 1996). To delineate the proper mechanism of action of the fraction, work will latter be directed towards using different phlogistic agents. This is, however, not within the premises of the present investigation. In conclusion, the aqueous ME of Alchornea cordifolia leaves could be beneficial in the management of different inflammatory disease states. The terpenoid fractions (A1, A2 and B) were found to be responsible for the antiinflammatory activity, together with the tannin-containing multi-component fraction (D). The analgesic activity exhibited by fraction A2 suggests an added advantage in the use of the extract as an anti-inflammatory agent, since most inflammatory conditions are usually associated with pains of varying degrees.

Acknowledgements We are ever grateful to Mr. Ozioko of Bioresource Development and Conservation Project of the same University, for authenticating the plant. Our thanks also go to Engr. (Dr.) N.N. Osadebe for his contributions during the statistical analysis.

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