Anti Nociceptive Studies Of The Root Bark Extracts Biology Essay

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Medicinal plants are believed to be an important source of new chemical substances with potential therapeutic effects [1,2]. The research into plants with alleged traditional use as pain relievers should be viewed as a fruitful and logical search for new analgesics drugs [2,3]. Maytenus senegalensis Lam Exell; locally called 'Bokaroro', 'Kunkushewa' or Namijin tsada', in Hausa; Tultulde or 'Yare-lesdi' in Fulani; 'Afor-juru' in Igbo and 'Sepolohun' in Yoruba languages [4] belongs to the family Celestraceae. It is a tall shrub with young branches often spiny, bearing leaves and flowers. Leaves appear grey, coriaceous, glabrous, very variable, obovate, oblanceolate or linear-spathulate, obtuse, entire or crenulate, decurrent at the base, 0.5-4.5 x 0.4-2 cm; petioles approx-imately 3 mm long. Flower appears small in axillary, dichotomous or fasciculate cymes on short branchlets, often forming terminal, elongate panicles. Pedicels filiform; bracts small, lanceolate, acute. Calyx lobes broadly elliptic-oblong, ciliate. Petals oblong. Disc fleshy, many lobed. Ovary glabrous, orbicular; style deeply 2-3-cleft. Capsules globose, 4-5 mm broad, bivalved, purple. Seeds chestnut brown; arillode orange. The plant flowers in October and found widely distributed in Tropical and subtropical Africa

and Asia. The root bark is used locally to treat dysentery [5]. An oil of some medicinal value is extracted from the seeds and known as oleum nigrum or Black oil [5]. An infusion of the roots are employed for healing wounds, ulcer and boils and also chipped into beer to serve as aphrodisiac while the leaves plus the roots is used for oedema [6]. The combination of the leave, root and bark is used for curing gastro intestinal disorder, dysentery, tooth decay and syphilis among others [6].

Materials and Methods

Plant Material

The root bark of the plant Maytenus senegalensis Lam Exell was collected around Yimirshika, Hawul - Borno State, Nigeria in the month of August 2006. The plant was identified by Dr. S. S. Sanusi of Department of Biological sciences and herbarium specimen with a herbarium number (12/006) was deposited at Natural Products Unit, Research Laboratory, Department of Chemistry, University of Maiduguri, Maiduguri -Nigeria.

Extraction of Plant Material

The plant material (root bark) was air-dried at room temperature for several days and pulverized with mortar and pestle. About 200g of the powdered root bark was extracted with 85% methanol using continuous Soxhlet extraction method to exhaustion. The extract was then concentrated in vacuo and a reddish-brown mass was obtained which weighed 34.5 g (17.25 % w/w) coded 'MSE'-Maytenus senegalensis extract.

Phytochemical Screening

The root bark extract was screened phytochemically for the presence of its constituents utilizing the following standard conventional protocols.

Tests for Alkaloids

Preliminary Test for Alkaloids: About 0.5 g of each extract was stirred with 5 ml of 1 % aqueous HCl on water bath and filtered. Of the filtrate, 3 ml was taken and divided equally into 3 portions in a test tube. To the first portion, few drops of Dragendorff's reagent was added, occurrence of orange-red precipitate was taken as positive. To the second 1 ml, Mayer's reagent was added and appearance of buff coloured precipitate was an indication of alkaloids and to the last 1 ml, few drops of Wagner's reagent was added and a dark-brown precipitate indicates the presence of alkaloids [7].

Confirmatory Test for Alkaloids: A confirmatory test designed to eliminate non-alkaloidal compounds capable of eliciting "false positive reaction" was carried out for the extract which gave a preliminary positive test for alkaloids. One gram of each extract was treated with 40 % calcium hydroxide until the extract was distinctly alkaline with litmus paper, and extracted twice with 10 ml portion of chloroform. The chloroform extracts were then combined and concentrated at reduced pressure to about 5 ml. This was then spotted on thin layer chromatographic (TLC) plates and developed using four solvents of different polarities. The presence of alkaloids in the developed chromatograms was detected by spraying with freshly prepared Dragendorff's spray reagent. A positive reaction indicated by the appearance of an orange or dark-coloured spots against a pale yellow background on the chromatograms was taken as confirmatory evidence that the extract contain alkaloid [8].

Test for Flavonoids

i. Shinoda's Test: About 0.5 g of the extract was dissolved in ethanol, warmed and then filtered. Three pieces of magnesium chips was then added to the filtrate followed by few drops of conc. HCl. A pink, orange, or red to purple colouration was an evidence for the presence of flavonoids [9].

ii. Ferric Chloride Test: About 0.5 g of the extract was boiled with water and then filtered. To 2 ml of the filtrate, few drops of 10 % ferric chloride solution were added. A green-blue, violet colouration indicates the presence of a phenolic hydroxyl group [10].

iii. Lead ethanoate Test: A small quantity of the extract were dissolved in water and filtered. To 5 ml of each of the filtrate, 3 ml of lead ethanoate solution was added. A buff coloured precipitate indicates the presence of flavonoids [7].

iv. Sodium hydroxide Test: About 2 ml of the filtered extract was dissolved in 10 % aqueous sodium hydroxide to give a yellow colouration. A change in colour from yellow to colourless on addition of dilute hydrochloric acid indicates the presence of flavonoids [10].

Test for Saponins Glycosides

About 1 g of the extract was boiled with 5 ml of distilled water, filtered and the filtrate divided into 2 portions:

To the first portion, about 3 ml of distilled water was added and then shaken for about 5 minutes. Frothing which persist on warming was an evidence for the presence of saponins. To the second portion, 2.5 ml of a mixture of equal volume of Fehling's solution A and B was added. A brick-red precipitate was taken as the presence of saponins glycosides [8, 11].

Test for Steroidal Glycosides

i. Liebermann-Burchard's Test (Steroidal nucleus): About 0.5 g of the extract was dissolved in 2 ml of acetic anhydride and cooled well in ice. Conc. tetraoxosulphate (VI) acid was carefully added. Thus, development of colour from violet to blue or bluish-green indicates the presence of a steroidal ring of the glycosides [12].

ii. Terpenoids: A little of the extract was dissolved in ethanol. To it 1 ml of acetic anhydride was added followed by the addition of conc. H2SO4. A colour change from pink to violet was an indication of the presence of terpenoids [12].

Test for Tannins

About 0.5 g of the extract was stirred with about 10 ml of distilled water and then filtered. The filtrate was used for the following test: To 2 ml of the filtrate, few drops of 1 % ferric chloride solution was added, occurrence of a blue-black, green or blue-green precipitate was taken as the presence of tannins.

A mixture of equal volume of 10 % lead ethanoate was added to 2 ml of the filtrate. Formation of a white precipitate indicates the presence of tannins. The filtrate of the extract was boiled with 3 drops of 10 % HCl and 1 drop of methanol, a red precipitate was taken as evidence for the presence of tannins [10,11].

Acute toxicity studies and analgesic effects.

Animals

A total of 43 adult male Swiss albino mice weighing between 18 and 27 g were obtained from Animal house, Department of Immunology, University of Maiduguri Teaching Hospital, Maiduguri; kept under well-ventilated conditions, fed on Standard feeds (Excel feeds Plc., Kaduna, Nigeria) and allowed water ad libitum.

Acute Toxicity Studies (LD50)

Acute Toxicity Studies (LD50) determination was conducted using the method described by Lorke (1983) [13]. In the initial phase mice were divided into 3 groups of three mice each and treated with methanol extract at doses of 10, 100 and 1000 mg kg-1 i.p and observed for 24 hours. In the final phase, 4 mice were divided in to 4 groups of one mouse each and methanolic extract administered at doses of 200, 400, 800, and 1600 mg kg-1 i.p and the final LD50 value calculated as described by Lorke, (1983) [13].

Acetic Acid-Induced Writhing (Anti-nociceptive studies)

This test was conducted employing the method described by Koster et al. (1959) [14] with little modification by Usman et al. (2005) [15]. Swiss albino mice were divided into 5 groups of 5 mice each. The first group served as negative control, (3 % gum acacia solution) 1 ml 100 g-1); earlier studies [15] have shown that gum acacia solution has no anti-nociceptive effects and that the extract under study dissolves well in it than does distilled water,

group 2 was given piroxicam at a dose of 20 mg kg-1 i.p. and served as positive control groups 3, 4 and 5 received the extract at doses of 25, 50 and 100 mg kg-1 i.p. 30 minutes later, all the groups were treated with acetic acid (0.07 %, l ml 100 g-1 i.p.). Mice were placed in individual cage. The numbers of abdominal constrictions were counted 5 minutes after acetic acid injection for a period of 10 minutes. Percentage inhibition of writhing was obtained using the formula: Inhibition (%) =

Inhibition (%) Mean Number of writhing (control) - Mean No. of writhing (test) x 100

Mean Number of writhing (control)

Vongtau, et al.(20); Usman et al. (15).

Statistical Analysis

All values were expressed as mean + SEM. Statistical analysis was carried out using the Student's t-test and differences between means were considered significant when P< 0.05.

Results

Phytochemical Screening

The Preliminary phytochemical analysis of the extract revealed the presence of alkaloids, flavonoids, glycosides, saponins, steroids and tannins as shown in Table 1.

Acute Toxicity Studies

The LD50 in mice of the methanolic root bark extract of Maytenus senegalensis was found to be 282.5 mg kg-1 body weight i.p. calculated by taking square root of the product of the dose of the survived group and lowest dose of the death group in the second phase of the acute toxicity study. That is the square root of 200 x 400 mg kg-1 body weight as described in the method used for this determination.

Anti-nociceptive Studies

The extract at doses between 50 and 100 mg kg-1 significantly (P <0.05) decreased the number of acetic acid-induced writhes as shown in Table 2 and Figures 1,2. Highest percent (82.9 %) inhibition was observed at dose of 100 mg kg-1 while that of the piroxicam 20 mg kg-1 was noted as 69.0 %. All values were significant (P< 0.05 and P< 0.01) compared with the negative control.

Discussion

The acute toxicity studies revealed that the extract had an i.p. LD50 of 282.5 mg kg-1 body weight i.p in mice, this indicates that the extract from this plant can be said to be moderately toxic

Table 1: Phytochemical analysis of the crude Methanolic root

bark extracts of Maytenus senegalensis

S/No

Constituents/ Test

Results

1.

ALKALOIDS

Dragendorff's Test

Mayer's Test

Wagner's Test

+

+

+

2.

CARBOHYDRATES

Molisch's Test

Barfoed's Test

Fehling (reducing sugar) Test

Fehling(combine reducing sugar) Test

+

+

+

+

3.

CARDIAC GLYCOSIDES

Legal's Test

Keller-killiani's Test

+

+

4.

FLAVONOIDS

Shinoda's Test

Lead acetate Test

NaOH Test

FeCl3 Test

+

+

+

+

5.

SAPONINS

Frothing Test

-

6.

STEROIDAL NUCLEUS

Salkowski Test

Liebermann - Burchard's Test

-

-

7.

TANNINS

FeCl3 Test

Lead acetate Test

+

+

+ = present, - = absent

Table 2: Effects of methanolic root bark extract of Maytenus senegalensis

on acetic acid (0.07% 1 ml 100 g-1) - induced writhing in mice

Treatment

Dose

(mg kg-1)

Number of

abdominal

constrictions

(mean ± SEM)

Inhibition

level (%)

P value

3 % gum acacia

solution (i.p.)

-

b23.4 ± 3.60

-

-

Extract (i.p.)

25

b22.5 ± 10.60

3.8

P>0.05

Extract (i.p.)

50

b16.5 ± 11.93

29.5

P>0.05

Extract (i.p.)

100

a4.00 ± 1.73

82.9

P<0.01

Piroxicam (i.p.)

20

a7.25 ± 5.68

69.0

P<0.01

Means with different superscripts are statistically significant

as the mice show general CNS depression restlessness without mortality. The methanolic root bark extract of Maytenus senegalensis significantly (P< 0.01) reduced the number of abdominal constriction induced by acetic acid in mice. Therefore, it could be suggested that there is a dose dependant activity since 100 mg kg-1 extract showed higher inhibition (82.9 %) relative to other doses and even proffer more protection than does piroxicam 20 mg kg-1 (69.0 %). Although this test is a non-specific model it is widely used for the evaluation of peripheral anti-nociceptive activity [16]. The method employed in this study called abdominal constriction response, is very sensitive and able to detect anti-nociceptive effects of compound(s) at dose level that may be inactive in other methods like tail-flick test [17,18]. The abdominal constrictions response is postulated to partly involve local peritoneal receptors [19]. The method has been associated with prostanoids in general, example increase level of PGE2 and PGF2α in peritoneal fluids [20,21] as well as lipoxygenase products by some researchers [22]. Therefore, owing to the fact that the extract under study is a flavonoid-rich as well as other phenolics and that analgesic and/or anti-inflammatory effects have been reported with flavonoids as well as tannins [15,23]. This result suggest that as the concentration of these plant bioactive components increases, so does protection against abdominal writhes induced by acetic acid. The results of the acetic acid-induced writhing strongly suggest that the mechanism of action of this extract may be linked to lipoxygenase and/or cyclo-oxygenases enzymes [21].

Conclusion

From the results obtained, it can be concluded that though moderately toxic, the plant extracts possess significant anti-nociceptive activity at relatively higher doses and thus, could be a view point for the use of the plant traditionally as a pain reliever. Study is underway to isolate and possibly characterize the bioactive phytochemical(s).

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