Anti-inflammatory study on crude methanol extract and different fractions of Eremostachys laciniata

Abstract

Context: Eremostachys laciniata (L.) Bunge (Lamiaceae), which has been reported as a rich source of flavonoids, is one of the rarely explored species of the genus Eremostachys.

Objective: In this study, the crude methanol extract and different fractions of E. laciniata were investigated for in vivo anti-inflammatory properties.

Material and methods: Shade-dried leaves of E. laciniata were exhaustively extracted by percolation with methanol (80%) to obtain 250 g of crude methanol extract (El), followed by fractionation with different organic solvents to get the n-hexane (Elh), chloroform (Elc), ethyl acetate (Ele), butanol (Elb), and water (Elw) fractions. An in vivo anti-inflammatory study of the crude extract and sub-crude fractions was carried out in rats using the carrageenan model.

Results: The Ele fraction was found to be the most potent inhibitor of edema formation by inducing a maximum inhibitory effect of 74.2% at the 300 mg/kg dose, during 3 h post carrageenan injection. The El extract and Elc fraction also showed good anti-inflammatory properties at the same dose.

Discussion: The demonstration of excellent anti-inflammatory activity by the plant chiefly concentrating in the Ele fraction and the appearance of peak activity in the latter phase of the experiment suggested the presence of relatively low-polar substances with arachidonic acid metabolite inhibition property.

Conclusion: The plant may be an excellent source in the future for activity-guided isolation of important anti-inflammatory substances.

Keywords::

• Rats
• paw
• carrageenan
• control
• inflammation

Introduction

When tissue injury occurs, whether caused by bacteria, trauma, chemicals, heat, or any other phenomenon, multiple substances are released by the injured tissues that cause dramatic secondary changes in the surrounding uninjured tissues. This entire complex of tissue change is called inflammation, which is characterized by vasodilatation of the local blood vessels, increased permeability of the capillaries, often clotting of the fluid in the interstitial space, migration of a large number of granulocytes and monocytes into the tissues, and swelling of the tissue cells (Guyton & Hall, 2006). Inflammation may be acute or chronic. Acute inflammation is short-lived, mainly involving neutrophils, whereas chronic inflammation can last for months or even longer and is characterized by the infiltration of macrophages and lymphocytes. Keeping in mind the importance of natural products in human healthcare, Eremostachys laciniata (L.) Bunge (Lamiaceae) was selected for anti-inflammatory study.

E. laciniata, a subalpine species, is a very close ally of E. speciosa Rupr. (Labiatae), regarded as a synonym for Phlomoides speciosa Rupr. (Adylov et al., 1986). It grows only in a natural environment at high altitude of about 2200 m, in sunny and sandy soil as well as shade. It has been reported from the eastern Mediterranean region, Central and Southwest Asia, Afghanistan, and Pakistan (Hedge, 1990; Jamzad et al., 2003; Stewart, 1862). The main constituents reported from the volatile oil of the plant included dodecanol (72.5%), widdrol (2.4%), germacrene B (1.9%), thujopsene (1.9%), and linalool (Navaei & Mirza, 2006). The plant has also been reported to possess potential antioxidant activity (Erdemoglu et al., 2006).

Material and methods

Plant material

Leaves of E. laciniata were collected in February 2002 at Chakdara, Malakand division of Pakistan. Identification was made by a plant taxonomist (Prof. Dr. Jehandar Shah, Islamia College, Peshawar) and a voucher specimen (EL-102) was submitted to the Botany Department, University of Peshawar.

Extraction and fractionation

Shade-dried leaves (4 kg) were powdered and exhaustively extracted with methanol (80%) by percolation at room temperature. The combined extract was dried using a rotary evaporator at ambient temperature to afford 250 g of crude methanol extract (El). Fractionation of El was carried out with different organic solvents to obtain n-hexane (Elh), chloroform (Elc), ethyl acetate (Ele), butanol (Elb), and water (Elw) fractions.

Animals

Sprague-Dawley rats of either sex, weighing 100–120 g, were used for the study. Animals were kept in a room maintained under environmentally controlled conditions of temperature 24 ± 1°C and 12 h light–12 h dark cycle. The animals had free access to both water and food. Experiments performed complied with the rulings of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (1996).

Carrageenan-induced rat paw edema

The method described by Winter et al. (1962) was used to test the crude and sub-crude fractions on acute inflammation. A freshly prepared carrageenan suspension 0.1 mL (1% in normal saline) was injected in the right hind paw of rats under the subplantar region. Edema volume was determined using a plethysmometer (Model 7150; Ugo Basile, Italy), prior to and 1, 3, and 5 h after carrageenan injection. Test samples were prepared by suspending the sample in normal saline, and given orally 1 h before carrageenan injection in doses of 100, 200, and 300 mg/kg. Positive controls were given aspirin as standard in a dose of 100 mg/kg orally 1 h before carrageenan injection, while negative controls received vehicle only.

Toxicity study

LD₅₀ values were assigned to the doses of crude extract and each sub-crude fraction that killed half of the test animals (Nayak et al., 2004).

Statistical analysis

The inhibition activity on carrageenan-induced rat paw edema caused by the oral administration of samples was measured and is reported as mean ± SEM. Statistical significance was determined using Student’s t-test (Olajide et al., 2004), and values of p < 0.001 and p < 0.01 were considered significantly different from control.

Results

The inhibitory effect of the crude methanol extract and sub-crude fractions of E. laciniata on carrageenan-induced increase in paw volume is shown in Table 1. Crude extract (El) exerted considerable control at 100 and 200 mg/kg doses on paw volume increase. Good control of increase in paw volume was observed with the 300 mg/kg dose of the same sample during 1 h post carrageenan injection, which was maintained throughout the experiment, with p ≤ 0.001. Excellent control of paw volume increase was observed throughout the experiment in groups treated with 300 mg/kg of the Ele fraction, with p ≤ 0.001. The same sample also showed good control of paw volume at the other doses used. The Elc fraction was found potent enough at the higher dose, i.e. 300 mg/kg, to control the increase in paw volume considerably for all stipulated durations, with p ≤ 0.01. A similar effect on increase in paw volume was observed for the rest of the groups and control, showing very low or no control of carrageenan-induced rat paw volume increase.

Table 1.  Effect of crude methanol extract and different fractions of E. laciniata on carrageenan-induced increase in paw volume.

Group	Dose (mg/kg)	Paw volume increase (mL)
		1 h	3 h	5 h
Control	—	0.34 ± 0.03	0.66 ± 0.03	0.72 ± 0.04
Aspirin	100	0.11 ± 0.04*	0.20 ± 0.03**	0.26 ± 0.04**
El	100	0.25 ± 0.02	0.37 ± 0.03*	0.44 ± 0.04*
	200	0.22 ± 0.03	0.30 ± 0.03**	0.36 ± 0.03
	300	0.16 ± 0.02*	0.28 ± 0.03*	0.30 ± 0.03*
Elh	100	0.33 ± 0.02**	0.65 ± 0.02	0.70 ± 0.03**
	200	0.33 ± 0.04*	0.66 ± 0.03**	0.66 ± 0.03*
	300	0.33 ± 0.04*	0.66 ± 0.04	0.66 ± 0.03*
Elc	100	0.22 ± 0.02*	0.43 ± 0.03*	0.48 ± 0.04**
	200	0.20 ± 0.03**	0.38 ± 0.03*	0.40 ± 0.04
	300	0.20 ± 0.02**	0.30 ± 0.03**	0.36 ± 0.02**
Ele	100	0.16 ± 0.03**	0.26 ± 0.04	0.32 ± 0.02*
	200	0.10 ± 0.02*	0.22 ± 0.04**	0.26 ± 0.03*
	300	0.10 ± 0.02*	0.17 ± 0.02*	0.20 ± 0.04*
Elb	100	0.33 ± 0.03*	0.60 ± 0.04	0.62 ± 0.02**
	200	0.32 ± 0.03	0.55 ± 0.04**	0.60 ± 0.04
	300	0.33 ± 0.02*	0.55 ± 0.03*	0.60 ± 0.04
Elw	100	0.30 ± 0.04**	0.61 ± 0.03**	0.70 ± 0.02*
	200	0.30 ± 0.02**	0.60 ± 0.03	0.70 ± 0.04
	300	0.30 ± 0.03**	0.60 ± 0.03	0.72 ± 0.04**

Note. Values are mean ± SEM (n = 6), *p ≤ 0.001, **p ≤ 0.01.

Looking at the plot of percent edema inhibition against varying doses of the different samples and standard illustrated in Figure 1, the crude extract (El) showed good inhibition of carrageenan-induced edema, i.e., 57.5 and 58.3% during 3 and 5 h at the 300 mg/kg dose. Similarly, the Elc fraction also showed a good inhibitory effect of 54.5 and 50% on edema formation with the same dose and durations. The Ele fraction was found to be the most potent inhibitor of edema formation at a dose of 300 mg/kg, showing excellent inhibition of carrageenan-induced edema throughout the experiment, i.e., 70.5, 74.2, and 72.2% at 1, 3, and 5 h, respectively. Other doses of the same sample also showed good activity for the stipulated durations.

Figure 1.  Inhibitory effect of increasing dose of methanol crude extract and different fractions of E. laciniata on carrageenan-induced edema.

Discussion

Carrageenan-induced rat paw edema has been commonly used for the study of acute inflammation, which is believed to be biphasic. The first phase (1–2 h) after carrageenan injection mainly involves inflammation mediated by histamine and serotonin and increased synthesis of prostaglandins in the surroundings of the damaged tissues. The later phase is sustained by prostaglandin release and mediated by bradykinin, leukotrienes, polymorphonuclear cells, and prostaglandins produced by tissue macrophages (Brito & Antonio, 1998). By comparing the inhibitory effect of the standard and samples on post carrageenan edema formation, it can be observed that both the El extract and Elc fraction showed slightly less activity than the standard at higher doses, whereas the Ele fraction was found to be an even better inhibitor of edema than the standard at a dose of 300 mg/kg during all observed durations. The peak inhibitory effect shown by El, Elc, and Ele samples at 3 and 5 h post carrageenan injection was quite similar to that exhibited by the group treated with aspirin. This inhibitory action of samples at the later phase of the experiment suggested the inhibition of arachidonic acid metabolites, which produce edema, dependent on neutrophil metabolism (Just et al., 1998). Further, the excellent anti-inflammatory activity demonstrated by the Ele fraction revealed the presence of relatively low-polar substances in the plant, with potential anti-inflammatory property. These observations may also add to the justification for the traditional use of the plant leaves as a poultice on bruises.

Conclusion

Considering these findings, it may be concluded that E. laciniata is a potential target for activity-guided isolation of important medicinal substances for the management and control of complications associated with inflammation.

Declaration of interest

The study was supported by grant from University of Peshawar.