Comparative phytochemical studies and anti-inflammatory activities of Desmodium velutinum ( Willd ) and Desmodium scorpiurus Desv . ( Family Papilionaceae ) growing in Nigeria

Members of the genus Desmodium are widespread in the sub-tropics, tropics and used in traditional medicine, as part of recipes for treating fever, pains, cough and dysentery. In this study, phytochemical screening and comparative anti-inflammatory activities of the methanol extracts, hexane and ethyl acetate fractions of the leaves of Desmodium velutinum and Desmodium scorpiurus were investigated using the egg-albumin induced oedema and protein denaturation methods. The presence of flavonoids, saponins, tannins and sterols were detected in the leaves of D. velutinum and D. scorpiurus while, the extracts and fractions exhibited anti-inflammatory activities not significantly different (p<0.05) from the activities of the reference drug (Diclofenac Sodium). The hexane fractions of D. velutinum and D. scorpiurus exhibited the most pronounced effects of 76.9 and 68.2% respectively at a dose of 2000 μg/ml in the in vitro model; 27.2 and 24.5% respectively in the in vivo model. This study has been able to report the anti-inflammatory activities of the leaves of D. velutinum and D. scorpiurus.


INTRODUCTION
The genus Desmodium is a member of the Papilionaceae family with more than three hundred and fifty (350) species widely spread and occurring throughout the subtropics and tropics (Lenne and Stanton, 1990).Traditionally, members of the genus Desmodium are used in the treatment of rheumatism, pyrexia, dysentery, cough, malaria and hepatitis while in the traditional Chinese medicine, most species of Desmodium are used in the treatment of fever, for neutralizing toxins, inhibiting pains and suppressing cough (Ma et al., 2011).
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License antipyretic analgesic and choleretic activities of Desmodium species (Liu et al., 2013) while, several flavonoids, alkaloids, terpenoids, steroids and a number of volatile oils have been characterized and isolated from about fifteen Desmodium species (Liu et al., 2013;Ma et al., 2011).However, most of the Desmodium species in Asia and Africa are yet to be fully studied in details.
D. velutinum is a perennial erect or sub-shrub which grows to about 3 m in height.It is used in folklore medicine for treating diarrhea, dysentery, stomachache (Setyowati-Indorto et al., 1999) and fever (Anowi et al., 2012) while, D. scorpiurus, commonly called Tick trefoil or Scorpion tick trefoil is a weak erect plant, used traditionally for the treatment of constipation, cough, convulsion, veneral infections and ringworm (Ndukwe et al., 2006).Though Desmodium velutinum and Desmodium scorpiurus are not documented for treating rheumatism and inflammation, the earlier reports of members of the genus Desmodium as part of recipes for treating inflammation (Liu et al., 2013) has led to investigating the anti-inflammatory activities and phytochemical analysis of these two species: D. velutinum and D. scorpiurus found growing in Nigeria.

Plant collection and authentication
The leaves of D.

Drying and extraction
The leaves of D. velutinum and D. scorpiurus were dried under shade and powdered in a miller.The powdered leaves were extracted by percolating in 80% methanol.They were filtered and the filtrates concentrated under reduced pressure to dryness after which a solvent-solvent partitioning was carried out successively with hexane and ethyl acetate to give the hexane and ethyl acetate fraction.

Phytochemical analysis
Phytochemical screening of the leaves of D. velutinum and D. scorpiurus were carried out using standard procedures (Evans, 2009).

Animals
Wistar albino male rats weighing between 130 and 150 g obtained from the Animal House of the University of Ibadan were allowed to acclimatize for two weeks before use and allowed12 h Light and 12 h dark cycle at 25°C.Animals were handled according to the International standard guiding the handling of animals and fed with standard pellets and allowed water ad libitum.

In vivo assay
The egg-albumin model was used in the in vivo anti-inflammatory study as the phlogistic agent (Anosike et al., 2012).The initial diameters of the right hind paws were taken and rats grouped into five groups (n=5).
Group A -Rats were administered 200 mg/kg of extract or fraction Group B -Rats received 100 mg/kg of extract or fraction.Group C-Rats were administered 50 mg/kg of extract or fraction.Group D -Rats were administered 5 mg/kg of Diclofenac (Cataphlam, Novartis) Group E-Rats were administered 1 ml/kg of distilled water.30 min after administering the extract, fraction or standard drug, 0.1 ml of egg albumin was injected into the right hind paw of each rat by sub plantar administration.Following the injection of the egg albumin, the hind paws of the rats were measured at an interval of 1 h for 6 h (0 to 6 h).The percentage inhibition of the edema was calculated as described by Jain and Khanna (1981).Io = Change in paw circumference in control group; I1 = change in paw circumference in treated groups.

In vitro anti-inflammatory assay
The in vitro anti-inflammatory assay was carried out using the method adopted from Sangita et al. (2012).0.2 ml of egg albumin (from fresh hen's egg) was added to 2.8 ml phosphate buffer saline (PBS, pH 6.4) and 2 mls of the extract/fraction or standard drug using five doses (2000, 1000, 500, 250 and 125 µg/ml) while, equal volume of distilled water served as control.The varying mixtures were incubated at 37±2°C for 15 min after which, the mixtures were heated in a temperature regulated water bath at 70°C for 5 min.After cooling, the absorbance of the mixtures was read in the spectrophotometer at 660 nm and the vehicle used as blank.The percentage inhibition of protein denaturation was calculated as:

Statistical analysis
The results are expressed as percentage inhibition (%) and data analyzed by using the one way analysis of variance (ANOVA) (p<0.05).

RESULTS AND DISCUSSION
The phytochemical screening of the leaves of D. velutinum and D. scorpiurus revealed the presence of flavonoids, saponins, tannins and sterols showing similarities in the secondary metabolites they possessed (Table 1).
The phytochemical analysis of D. velutinum used in this study is however similar to the one collected from the South east region of Nigeria (Anowi et al., 2012).Furthermore, there were similarities in the secondary metabolites of the leaves of D. scorpiurus and D. velutinum as well as those Desmodium species previously studied (Ma et al., 2011;Liu et al., 2013).
The egg-albumin model used in this study is one of the models adopted for determining anti-inflammatory and anti-oedematogenic activities of medicinal plants (Hira et al., 2013).The model induces inflammation by producing histamine, serotonin and bradykinin in the first phase and the release of prostaglandins and nitric oxide in the second phase by showing peak inflammaton at 3 h (Seibert et al.,1994).
The methanol extracts, hexane and ethyl acetate fractions of D. velutinum and D. scorpiurus were investigated for their anti-inflammatory potentials.In the in vivo anti-inflammatory study, the activities of the methanol extract of D. velutinum leaf at 200 and 100 mg/ml were not significantly different (p>0.05) from each other but more pronounced than that of the reference drug (Diclofenac sodium) when compared with the untreated group.The methanol extract at 200 and 100 mg/ml exhibited a reduction in oedema of 28.8 and 32.1% at 2 h respectively while, the hexane fraction of the leaf of D. veluti num at 200 and 100 mg/kg exhibited same anti-inflammatory effect by reducing the oedema by 27.2% at 6 h (Tables 2 and 3).The ethyl acetate fraction exhibited a weak anti-inflammatory effect of 13.2 and 6.8% at 200 and 100 mg/ml after 6 h (Table 4).
The methanol extract of the leaf of D. scorpuirus exhibited dose dependent anti-inflammatory activities at all doses (200, 100 and 50 mg/ml) tested.The methanol extract at 200 and 100 mg/ml exhibited high antiinflammatory effects of 27.1 and 21.9% 6 h after extract administration when compared to the untreated group (Table 5).The hexane fraction 6 h after administration at 200 and 100 mg/kg exhibited a reduction of 24.5 and 18.1% in the oedema and the activity of the ethylacetate was the weakest with a reduction of 18.1 and 14.3% at 6 h.This effect is similar with that exhibited by the ethylacetate fraction of the leaf of D. velutinum.In the in vitro assay, the methanol extracts, hexane and ethyl acetate fractions of D. velutinum and D. scorpiurus exhibited dose-dependent activities.
This study shows that the anti-inflammatory activities exhibited by the leaves of D. velutinum and D. scorpiurus are mainly in the hexane fraction, and could probably be due to non-polar compounds present in the leaves.In addition, the anti-inflammatory activities exhibited by the leaves of D. velutinum and D. scorpiurus are similar to some other species of Desmodium previously reported: D. gangeticum (Govindarajan et al., 2001) and D. triflorum (Lai et al., 2010).The methanol extracts and fractions of D. velutinum and D. scorpiurus lowered the paw oedema from the 2nd to 6th h (Tables 6 and 7).This is similar to the effect of the methanol extract of D. triflorum at the 3rd to 6th h at doses of 0.5 and 1 g/kg (Shang-Chih et al., 2009).The activities of the extracts and fractions of D. velutinum and D. scorpiurus were similar to the effect of the reference drug used, (Diclofenac Sodium) which reduces inflammation by inhibiting the synthesis of prostaglandin (Della et al., 1986) through inhibition of the cyclooxygenase in the arachidonic acid pathways (Skoutakis et al., 1988;Chen et al.,1995).
The activities of flavonoids, tannins, sterols and saponins present in the leaves of D. velutinum and D. scorpiurus have been attributed to anti-inflammatory activities, and previous studies have reported the potentials of flavonoids and other phenolic compounds in exerting anti-inflammatory activities by inhibiting the enzymes involved in the mediators of inflammation (Sawadogo et al., 2006) or by inhibiting the migration of leukocyte, reducing serum lysozyme levels and nitric acid (Wu et al., 2006).
The potentials of the leaf extracts and fractions of D. velutinum and D. scorpiurus to reduce paw oedema from the second hour shows the ability of the compound(s)   in the leaves to inhibit production of serotonin and bradykinnin in the first phase as well as inhibit the release of prostalglandins and nitric oxide responsible in the second phase of inflammation.

Conclusion
This study has therefore been able to further justify and report the anti-inflammatory effect of some other members of the genus Desmodium apart from those previously studied.
velutinum and D. scorpiurus were collected from Eruwa near Ibadan in Oyo State in August, 2012 and authenticated at the Forestry Research Institute of Nigeria by Mr Chukwudi where, voucher specimens with voucher numbers FHI 108953 and 109646 were deposited.

Figure 2 .
Figure 2. Effect of the methanol extract, hexane and ethyl acetate fractions of D. scorpiurus leaf against protein denaturation.

Table 2 .
In vivo anti-inflammatory activities of methanol extract of D. velutinum leaf in egg albumin induced rats.
Percentage decrease of anti-inflammatory effects compared to the untreated group,*p<0.05. Figure 1.Effect of the methanol extract, hexane and ethyl acetate fractions of D. velutinum leaf against protein denaturation.

Table 3 .
In vivo anti-inflammatory activities of hexane extract of D. velutinum leaf in egg-albumin induced rats.decrease of anti-inflammatory effects compared to the untreated group, *p<0.05. Percentage