Anti-Inflammatory, Anti-pyretic and Acute Toxicity Effects of n-Butanol Extract of Atractylis flava Desf in Rats

Plant material

The whole plant Atractylis flava Desf was collected from Biskra Algeria in the month of May 2015. The plant materiel was identified by Prof. Bachir Oudjehih (Agronomic Institute of Batna1 University, Algeria). A voucher specimen number (660/LCCE) was deposited in the herbarium of the mentioned department.

Animals

Experiments were performed using Wistar rats of both sexes, weighing (150 – 180 g). The animals were obtained from the Pasteur institute, Algiers, Algeria. The animals were kept in polypropylene cages under standardized conditions for 8 days before experiments. The animals were fed with standard diet and water ad libitum.

Extraction

The collected whole plant Atractylis flava was air-dried and powdered. 500 g powder was macerated with MetOH-H₂O (80:20). After filtration, the filtrate was concentrated under vacuum at room temperature the hydro alcoholic extract was submitted to liquid-liquid fractioning using solvents with increasing polarities (petroleum ether, dichloromethane, ethyl acetate, and n-butanol). The n-butanol fraction was used for the investigation of the acute toxicity study, anti-inflammatory and antipyretic effects.

Acute toxicity study of the extract

Acute oral toxicity study was performed as per the OECD-423 guidelines.¹⁰ Ten Wistar female rats were used for this study; they were divided into two groups of five animals each. The animals were fasted overnight and allowed free access to water, after A. flava n-butanol extract was administered orally in a single dose of 2000 mg/kg body weight. Animals were observed individually after dosing at least once every 30 min, periodically during the first 4 h, with special attention given during the first 24 h.

General symptoms of toxicity and mortality in each group were observed within 24 h. Animals that survived after 24 h were observed daily for any signs of delayed toxicity for two weeks. At the end of 14 days observation period, the animals were anaesthetized, and blood samples were collected through cardiac puncture with and without anticoagulant (EDTA) for hematological and biochemical analysis, respectively.

Egg albumin induced paw edema in rats

The anti-inflammatory activity of the tested extract was evaluated in male Wistar rats by the egg albumin induced paw edema method.¹¹ the animals were divided in five groups (n = 5).

All groups were kept fasting and allowed free access to water.

Group I was treated orally with normal saline (10 mL/kg), group II with diclofenac (30 mg/kg), the rest of groups (III, IV and V) were treated with Atractylis flava n-butanol extract at the dose level of 100, 250 and 500 mg/kg p. o. After 1 h, inflammation induced by sub plantar injection of 100μL of undiluted fresh egg albumin into the right hind paw of all rats. The paw thickness of each rat of all groups was measured in mm using digital vernier calipers at 0, 1, 2, 3, and 5 h.

The percentage inhibition of edema by the tested extract and standard drug was calculated in comparison with vehicle control using this following formula:

Where tCn = paw thickness at particular time point of control animal; tC0 = paw thickness before induction; tTn = paw thickness at particular time point of treated animal; and tT0 = paw thickness before induction.

Brewer’s yeast induced pyrexia

Antipyretic activity was measured by Brewer’s induced pyrexia in male Wistar rats.¹² Rats were fasted overnight with free access to water before the experiments. Pyrexia was induced in animals by subcutaneous injection of 20 % brewer’s yeast (10 mL/kg) suspended in saline solution into back side of below the nape of the neck. After 17 h of yeast injection, the rectal temperature of each rat was measured using a digital thermometer. Only rats that showed rectal temperature of 38°C and above were selected for the experiments. Rats were divided into five groups. The 1^st group was kept as a control (received the normal saline) while the 2^nd one was given paracetamol in a dose of 150 mg/kg (standard). The 3^rd - 5^th groups received orally 100, 250 and 500 mg/kg of the BEAF, and the rectal temperature was measured periodically at 1, 2, 3 and 5 h after drug administration.

Statistical analysis

Data were expressed as the mean ± SEM. The data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Differences between two means were detected using the Student’s t-test (acute toxicity test). Data were considered different at significance level of p < 0.05.

Acute toxicity study

The acute toxicity test revealed that the oral administration of a single dose (2000 mg/kg) of BEAF to rats did not bring any signs of toxicity or mortality in treated animals during the 14 days observation period. In addition, there was no significant difference in hematological and biochemical parameters between control and treatment group (Table 1 and 2). This indicates that BEAF was nontoxic in rats up to the dose of 2000 mg/kg of body weight (Table 1 and Table 2).

Effect of the BEAF on egg albumin induced paw edema in rats

The percentage rate of effect of BEAF in fresh egg albumin induced rat paw edema test and the average edema for the various groups was demonstrated in (Table 3). Pre-treatment with BEAF exhibited significant anti-inflammatory activity during various assessment times (1-5 h) in a dose dependent manner. The highest inhibition of edema was obtained with 500 mg/kg dose (57.54%) at the 5^th hour of drug administration. (Table 3).

Table 1:

Biochemical parameters of control and treated rats; measured during the acute toxicity study.

Biochemical parameters	Control	BEAF 2000 mg/kg BW
Liver profile
AST(U/L)	131 ± 5.54	122.8 ± 3.36
ALT(U/L)	86.2 ± 7.13	78.4 ± 9.66
Total bilirubin(mg/dL)	0.76 ± 0.04	0.70 ± 0.07
Renal profile
Urea (mg/L)	0.30 ± 0.01	030 ± 0.01
Creatinine (mg/L)	4 ± 0.01	3 ± 0.0
Lipid profile
Cholesterol (mmol/L)	0.57 ± 0.04	0.63 ± 0.05
Triglycerides (mmol/L)	0.83 ± 0.10	1.14 ± 0.15
HDL-Cholesterol (mmol/L)	0.50 ± 0.03	0.54 ± 0.06
LDL-Cholesterol (mmol/L)	0.06 ± 0.01	0.06 ± 0.01
Glucose (g/L)	0.67 ± 0.03	0.68 ± 0.05

[i] Values are mean±SEM(n=5). No significant compared to control.

Table 2:

Hematological parameters of control and treated rats; measured during the acute toxicity study.

Hematology parameters measured	Control	BEAF 2000 mg/kg BW
RBC(106xμL)	7.35±0.14	7.13±0.06
MCV(fL)	55.68±0.85	57.06±0.67
RDW(fL)	13.18±0.26	13.22±0.25
HCT (%)	40.92±0.48	40.74±0.42
PLT (103/μL)	614.8±61.76	645.4±50.75
MPV (fL)	6.18±0.09	6.32±0.13
WBC (103/μL)	6.10±0.69	6.92±1.10
HGB(g/dL)	14.5±0.17	14.34±0.11
MCH(pg)	19.74±0.25	20.08±0.22
MCHC(g/dL)	35.46±0.17	35.22±0.13
Lymphocyte no	4.24±0.51	4.82±0.93
Lymphocyte %	69.08±2.43	68.76±2.73

[i] Values are mean±SEM(n=5). No significant compared to control.

Table 3:

Effect of the n-butanol extract of A. flava (BEAF) on egg albumin-induced paw edema in rats.

Treatment Dose (mg/kg)	Δ paw edema (m m) and % inhibition
	1h	2h	3h	4h	5h
Normal saline10(mL/kg)	5.43±0.11	4.92±0.21	4.64±0.15	4.09±0.17	3.80±0.13
BEAF 100	5.19±0.11 (4.42%)	4.46±0.21 (9.50%)	3.83±0.16** (17.49%)	2.78±0.11*** (31.83%)	2.35±0.10*** (38.12%)
BEAF 250	5.02±0.09 (7.53%)	4.28±0.19 (13.07%)	3.66±0.15*** (21.11%)	2.57±0.10*** (36.96%)	1.79±0.06*** (52.67%)
BEAF 500	4.76±0.10** (12.40%)	3.89±0.17** (20.93%)	3.18±0.08*** (31.37%)	2.25±0.087*** (44.59%)	1.61±0.05*** (57.54%)
Dichlofenac 30	4.85±0.07** (10.65%)	3.87±0.16** (21.43%)	2.71±0.08*** (41.59%)	1.94±0.07*** (52.31%)	1.24±0.03*** (67.28%)

[i] Values are mean±SEM(n=5). *P<0.05, **P<0.01, ***P<0.001 compared to control.

Figure 1:

Percent of the antipyretic effect of BEAF in Brewer’s yeast induced pyrexia test; [A]paracetamol, 150mg/kg [B] 500mg/kg and [C] 250 [D] 100 mg/kg. Values are reported as mean ± S.E.M (n=5). *P<0.05, **P<0.01, ***P<0.001 compared to control.

DISCUSSION

Plants exhibit various pharmacological activities because of the presence of diverse kinds of constituents. It is compelling evidence that many chronic diseases such as inflammatory disorders, rheumatism, diabetes, cardiovascular and many neurodegenerative disorders have been treated by plants or their bioactive compounds used in the traditional medicine those can be a new therapeutic source for the treatment of chronic dis-orders.¹³

The safety studies on medicinal plants were conducted by performing acute and sub-acute toxicity tests in laboratory animals.¹⁴ Acute toxic-ity study showed that the n-butanol extract of Atractylis flava Desf possessed high safety profile, as BEAF did not present noticeable signs of toxicity or mortality at the dose of 2000 mg/kg, in any animal during the entire observation period. No significant differences on hematological and biochemical parameters when compared to the control group. These results indicated high margins of safety and explain the traditional use of the plant.

Egg albumin induced rat paw edema is one of a commonly used primary test to evaluate the anti-inflammatory effect of natural products. Edema formation results from the synergistic action of inflammatory mediators such as histamine, serotonin and bradykinin produced under the effect of cycloxygenase-2 (COX-2) at the site of a local inflammatory insult leading to increased vascular permeability and blood flow.^15,16 Edema formation due to egg albumin in the rat paw is a biphasic event; the early phase of edema, which begins immediately after the administration of the irritant and lasting up to 2 h, is probably due to the release of histamine and serotonin. While the latter phase, occurring from 3 to 5 h after the administration of the irritant is induced by bradykinin, protease, prostaglandins and lysosome.¹⁷

The results of this study showed that BEAF significantly inhibited the formation of the paw edema during the first phase and significantly maintained in the second one. It can be suggested that this anti-inflammatory effect probably might be attributed to the inhibition of the release of pro-inflammatory mediators of acute inflammation, especially the prostaglandins.

Fever is a surrogate marker for disease activity in many infectious and inflammatory disorders. According to the classical view, the genesis of fever is induced by inflammatory mediators (i.e., cytokines, namely interleukin-1, interleukin-6, tumor necrosis factor, and others) that are predominantly released by activated peripheral mononuclear phagocytes and other immune cells.^18,19 Antipyretic activity is commonly considered as a feature of drugs or compound which have an inhibitory effect on the biosynthesis or release of prostaglandins.²⁰

The subcutaneous injection of brewer’s yeast evoked pyrexia by ultimately increasing synthesis of prostaglandin and is considered as a valuable i« vivo screening test for the assessment of antipyretic potential.^21,22,23 The inhibition of prostaglandin synthesis could be the possible mechanism of antipyretic action as that of paracetamol and the inhibition of prostaglandin can be achieved by blocking the cyclooxygenase enzyme activity.^24,25,12 The antipyretic effect of BEAF may be due to inhibiting the enzyme cyclooxygenase and reducing the level of prostaglandin within the hypothalamus in pyrexia rats.

Phytochemical study of the whole plant Atractylis flava showed abundance of flavonoids triterpenes, steroids, and saponins. Furthermore, the presence of a tiliroside, narcissin, vicenin 3, ladaneine and schaftoside were detected in the «-butanol extract.^{8, 9}

Flavonoids have been demonstrated that they are able to inhibit a series of enzymes, which are activated in the course of the inflammatory process.²⁶ Also, flavonoid such as vicenin-2, Tiliroside, Schaftoside, have been recognized as potent inhibitors of pro-inflammatory mediators in different studies.^27,28,29,30 Anti-inflammatory and antipyretic action of BEAF may be due to the presence of above phytoconstituents. Hence, the effect may be due to the synergistic effect or than single constituent.