Phytochemical and Antisickling Studies of the Leaves of Hymenocardia acida Tul (Euphorbiaceae)

MATERIALS AND METHODS

Plant collection and preparation: The branches of the Hymenocardia acida were collected from bushes of Dajin Tohu around Shika Dam, Zaria. It was identified on the field using descriptions given in the monograph (Dalziel, 1937; Keay et al., 1964). It was authenticated at the Herbarium, Biological Sciences, Ahmadu Bello University (A.B.U), Zaria. The voucher specimen number given was (7108) identified by A.O. Ohieri.

The leaves of H. acida were separated from the branches, dried under the shade for 2 weeks and then in the oven at 40°C for 24 h. The dried leaves were powdered using mortar and pestle and sieved using sieve mesh size 20.

The following studies were conducted at the Department of Pharmacognosy and Drug Development, A.B.U, Zaria. Nigeria.

Phytochemical screening: The leaves were tested for the presence of chemical constituents such as carbohydrates, tannins, resins, balsams, glycosides, terpenes and steroids (Evans, 1996; Balbaa, 1976; Sofowora, 1993).

Scheme I:	Extraction of the leaves of Hymenocardia acida. Key: Pet. Ether-Petroleum ether, H2O-Water, Et. OH-Ethanol, n-BuOH-n-butanol, HCl-hydrochloric acid

Extraction of the leaves of H. acida: Fifty grams of the powdered leaves was extracted by maceration with ethanol. The extract was concentrated in vacuo and used to test for the antisickling activity.

The fractionation was carried out as given on scheme I (Woo et al., 1980). Fractions B, 1, 2, 3 and C resulting from the ethanolic extracts were tested for antisickling activity. The residues were dissolved in water or their respective solvents used for fractionations.

Antisickling activity: The antisickling activity (Reversal of sickled RBC) was determined using standard methods (Sofowora, 1979; Fasanmade; Olaniyi, 1991).

RESULTS

Phytochemical screening: The phytochemical screening revealed the presence of carbohydrates, tannins, flavonoids, saponins, cardiac glycosides. terpenes, steroids and alkaloids in the leaves of H. acida.

The chemical test of the various fractions revealed the following compounds A-terpenes, steroids and alkaloids; B-carboxylic acid; 1-Saponins and flavonoids; 2-saponins; 3-saponins; C-flavonoids.

Antisickling activity: The leaves were found to possess antisickling activity. The activity was found to be dose dependent (Table 1-4). The RBC were observed to change from the sickled shape to normal biconcave cells and later observed to increase in size after 30 min (Table 2-4).

All the various fractions (B, 1, 2, 3, C) soluble in water were observed to reverse sickling. The ether soluble fraction causes deformation of the cells. They were seen

Table 1:	Inhibition of sickling-leaves of H. acida
Key - Absence of sickled cells, Average values in bracket, %w/v Percentage weight by volume, min minutes

Table 2:	Results of effect of the plant ethanol extract on erythrocyte sickling at 2% concentration

Table 3:	The effect of plant ethanol extract on erythrocyte sickling at 1% concentration

Table 4:	The effect of plant ethanol extract on erythrocyte sickling at 0.5% concentration

to be irregularly shaped and smaller in size. The clusters of RBC observed with ethanol extract were not observed with the fractions.

The ethanolic extracts of the leaves were seen to reverse the sickling of RBC. Many drugs given to patients in the management of sickle cell conditions in our hospitals do not have effect on the sickle cells. The treatments are usually supportive with haematenics (Sofowora, 1979), analgesics and fluid infusion. Some plants have been discovered to have antisickling activity like the Fagara zanthoxyloides (Sofowora, 1979). F. zanthoxyloides is not available in northern part of Nigeria. Therefore the discovery of other plants with antisickling activity will supplement it.

The study revealed that the leaves of H. acida contained various chemical constituents which led to the fractionation of the plant leaves extract.

All the fractions tested (B, 1, 2, 3 and C) were found to reverse the sickling of RBC. Therefore carboxylic acids, flavonoids and saponins are responsible for the antisickling activity of the leaves of H. acida. The fractions did not produce clusters of reversal sickled cells as with the ethanol extracts. This shows the purification and isolation will enhance the antisickling activity of the constituents. Saponins have been shown to possess antisickling activity (Evans, 1996; Moody et al., 2003). Zanthoxylol, a butyric acid derivative and 1-hydroxylbenznoic acid were isolated from Fagara zanthoxyloides which are responsible for the antisickling activity of this plant (Sofowora, 1979). This research has shown that more than one constituent is responsible for the antisickling activity. Isolation of anyone of them can be used to formulate a standard preparation. Scientific evidence has been provided for the use of this plant in the management of sickle cell anaemia.

Further work needs to be done to isolate and characterize the various compounds responsible for the antisickling activity.

The leaves were found to contain carbohydrates, tannins, glycosides (saponins flavonoids), terpenes, alkaloids and steroids. Also, it could reverse sickled red blood cells. The fractions containing saponins, flavonoids and carboxylic acid were found to be responsible for this activity. Therefore, from the above results; the plant Hymenocardia acida might be potential sources of antisickling agent for sickle cell patients.

This justifies the use of plant in the management of sickle cell disease.

We wish to acknowledge Mr. Peters of Haematology Department, Ahmadu Bello University Teaching Hospital, Zaria, Nigeria for assisting us with blood sample. Also, Adamu Mohammed of the Department of Pharmacognosy and Drug Development, Ahmadu Bello University, Zaria, Nigeria who is a laboratory assistant.