Anti-inflammatory activities of the methanol leaf extract and fractions of Elaeis guineensis in in vivo animals

Abstract

Objectives

The objective of this paper is to investigate the preliminary anti-inflammatory properties of the leaves and its fractions of Elaeis guineensis using egg-induced albumin and formaldehyde-induced arthritis models in rats.

Methods

Egg albumin-induced inflammation was induced by sub-plantar injection of 0.1 ml of fresh undiluted egg albumin. The volume of distilled water displaced by the treated paw was measured at 0.5,1, 2, 3, 4, 5, and 6 h after induction of inflammation. Formaldehyde-induced arthritis was induced after 30 min by sub-plantar injection of 0.1 ml of 2.5% formaldehyde solution and was repeated on Day 3. Arthritis was assessed by measuring the volume of distilled water displaced by the paw before induction of arthritis and once every day for 10 days, starting from Day 1, after induction of arthritis.

Key findings

The acute toxicity test revealed that the plant extract was safe even at 5000 mg/kg dose level. The methanol extract showed a dose-dependent inhibition of egg albumin-induced acute inflammation that peaked at the dose of 200 mg/kg, while the fractions of the extract showed that the hexane and ethyl acetate fractions had the highest activity during the experiment. Similarly, the crude extract of the formaldehyde-induced arthritis showed a dose-dependent inhibition making the 200 mg/kg the most active dose level. In addition, it was observed that among the fractions of the extract, the hexane fraction showed the most significant inhibitory activity when compared to other fractions.

Conclusions

This study establishes that the methanol leaf extract and fractions of E. guineensis possess significant anti-inflammatory activity.

Introduction

The body’s immune system naturally and defensively reacts to damage, infection, or irritation by producing inflammation. The goal of this intricate biological process is to eliminate damaging stimuli and start the healing process by interacting with different immune cells, signalling molecules, and tissues. It is characterized by vasodilation and increased permeability, immune cell activation, inflammatory mediators, resolution, and tissue repair [1].

There are two types of inflammation that are acute and chronic inflammation. Acute inflammation is characterized by the classic symptoms of vascular and exudative processes predominating in acute inflammation [2]. It is the body’s initial reaction to harmful stimuli caused by the penetration of blood plasma and leukocytes into injured tissue. Acute inflammation is categorized histologically by a complex series of events that include vasodilation of the blood vessels, resulting in increased local blood flow, increased capillary permeability, resulting in fluid and blood leakage into the interstitial space, clotting of the fluids in the interstitial space due to excess fibrinogen leaking from the capillaries, and leukocyte migration [3]. The cardinal symptoms of acute inflammation include pain, redness, immobility, swelling, and heat [4].

Chronic inflammation is also a type of acute inflammation that lasts longer than normal, is more persistent, and is typically brought on by autoimmune responses, persistent foreign bodies, and nondegradable microorganisms. Acute inflammation can lead to chronic inflammation, as in the case of pneumonia, as well as without acute inflammation, as in the case of tuberculosis, viral infections, and rheumatoid arthritis. In most cases, it is irrevocable [5].

Natural plant-based products have been used for medical purposes since ancient times. This practice is ingrained in traditional healing methods seen in many different cultures across the globe.

For millennia, people have understood and utilized the medicinal properties of plants, from traditional cures to contemporary medications.

Natural medicines have seen a rise in popularity recently because of their apparent efficacy, low side effects, and ecological sustainability. A wide range of botanicals, including herbs, roots, flowers, fruits, and leaves, are included in the category of ‘natural plant products’. Each of these materials has a special combination of bioactive components with therapeutic qualities. These substances have a variety of physiological effects on the human body, including analgesic, immunomodulatory, anti-inflammatory, and antibacterial properties.

The herbal plant Elaeis guineensis has been reported by previous scientists because of its medicinal value and anti-inflammatory properties. According to Ref. [6], the leaf of E. guineensis contains flavonoids and other phenolic compounds that possess anti-inflammatory activities. In several animal studies, the extracts of E. guineensis leaves have been shown to reduce inflammation and oxidative stress [7] and reported also its acute anti-inflammatory healing properties in mice.

Materials and methods

Chemicals

Analytical-grade materials and reagents were utilized in the investigation. These consist of the following: methanol, n-hexane, ethyl acetate, Mayer’s reagent, Dragendorf’s reagent, Wagner’s reagent, 20% potassium hydroxide solution, ferric chloride solution, lead acetate solution, 1% aluminium chloride solution, acetone, concentrated hydrochloric acid, Millon’s reagent, concentrated nitric acid, dilute sodium hydroxide, and copper sulfate crystals. All of these products are supplied by Sigma-Aldrich in Germany. The supplier of formaldehyde was Caldic in Germany, and the source of the eggs was a nearby Nsukka store.

Plant material

In March 2021, fresh palm fronds, or E. guineensis, were gathered from Orba, Nsukka L.G.A. in Enugu State. Mr. Alfred Ozioko, a plant taxonomist of the International Centre for Ethnomedicine and Drug Development Nsukka, completed the official identification. After 28 days of air drying at room temperature, the leaves were milled into a fine powder.

Animals

The study used mature Wistar rats (120–200 g) of both sexes and Swiss albino mice (26–34 g). Every animal was acquired from the University of Nigeria Nsukka’s Department of Pharmacology and Toxicology. They had unrestricted access to water and were kept in cages made of stainless steel.

They were fed regular feed pellets. The research was carried out in compliance with the updated National Institute of Health Guide for Care and Use of Laboratory Animals (Pub No. 85-23, revised 1985) as well as the ethical guidelines and recommendations of the University of Nigeria committee on the care and use of laboratory animals.

Acute toxicity study

To determine the range of the plant extract’s lethal and safe doses, an acute toxicity test was conducted using the Lorke method. For the study, 18 Swiss albino mice were given food for 18 hwhile being able to access water. The mice were then divided into three groups and given varying doses of the plant extract (10, 100, and 1000 mg/kg) intraperitoneally (i.p.). The animals were then monitored for signs of nervousness, dullness, incoordination, and mortality for 24 h.

Anti-inflammatory test

Two methods were used to assay the anti-inflammatory activity of E. guineensis. Twenty-five adult Wistar rats of either sex (120–200 g) were randomly divided into five groups of five rats each was used for each method. They were fasted and deprived of water for 18 h before the experiment.

Egg albumin-induced acute inflammation

The rat paw oedema method of Okoli and Akah [8] was used as modified. All the treatments were administered orally. Twenty-five rats were randomly divided into five groups. Group I (negative control) received (10% Tween 80), group II (positive control) received 10 mg/kg indomethacin, while groups III, IV, and V received 100, 200, and 400 mg/kg of ME, respectively. Thirty minutes after administration, inflammation was induced by sub-plantar injection of 0.1 ml of fresh undiluted egg albumin. This was obtained by carefully extracting the white portion of egg contents from the yolk (yellow portion) of the egg, using a 1-ml syringe. The volume of distilled water displaced by the treated paw was measured at 0.5,1, 2, 3, 4, 5, and 6 h after induction of inflammation. The fractions were later screened using the same procedure but at the most active dose level of the ME.

In each case, the level of inhibition of oedema in the animal paws was calculated relative to the negative control at periods 0.5–6 h using the relation [8]:

where, a is the mean paw oedema volume of treated animals after egg albumin injection; x is the mean paw oedema volume of treated animals before egg albumin injection; b is the mean paw oedema volume of control animals after egg albumin injection; and y is the mean paw oedema volume of control animals before egg albumin injection.

Formaldehyde-induced arthritis

This experiment was done according to the method of Seyle (1949) was used as modified by Okoli [9]. Adult male albino rats (120–200 g) were divided into five groups (n = 5). The basal right hind paw volume was measured by the volume displacement method. On Day 1 of the experiment, group 1 (negative control) was treated with Tween 80 (10%) group II (positive control) was treated with indomethacin (10 mg/kg), while groups III, IV, and V were treated with the crude extract at 100, 200, and 400 mg/kg, respectively—all by the oral route. Inflammation was induced after 30 min by sub-plantar injection of 0.1 ml of 2.5% formaldehyde solution and repeated on Day 3. Arthritis was assessed by measuring the volume of distilled water displaced by the paw before induction of arthritis and once every day for 10 days, starting from Day 1, after induction of arthritis. Extract administration was continued once daily for 10 days. The fractions were later screened using the same procedure but at the most active dose level of the ME. The level of inhibition of arthritis was calculated using the relation:

where V_c is the mean oedema in control, andV_t is the mean oedema in the group treated with standard/extract.

Statistical analysis

Graph Pad Prism 8.42 was used to analyse the data, and Dunnett’s post hoc test was applied. The findings were presented as mean ± standard error of the mean, with P < .05, .01, .001, and .0001 denoting significant differences in the means of the treatment and control groups.

Results

Yields from extraction and fractionation

Elaeis guineensisYield (w/w)

The weights of the extract during the various aspects of the extraction process and the associated yields from the extraction and fractionation processes are outlined below:

The crude extracts (g)

$W2(weightofdriedpowderedplantmaterial$

$beforeextraction)=1341g$

Fractions

$Methanolfraction(MF)44.12×100/91=48.5%$

$Hexanefraction(HF)5.82×100/91=6.4%$

$Ethylacetatefraction(EF)14.01×100/91=15.4%$

Phytochemical screening

Table 1 shows the results of the phytochemical screening of the crude extract and its fractions. The findings indicate that alkaloids, glycosides, flavonoids, tannins, steroids, and saponins are present in the crude extract of E. guineensis, with saponins being the most prevalent phytoconstituents and glycosides the least. But out of all the fractions, the MF had more alkaloids and flavonoids than saponins or steroids, while the hexane fraction (HF) had more steroids and resins than glycosides. However, the ethyl acetate fraction (EF) contained no steroids and very modest levels of tannins, alkaloids, flavonoids, resins, and saponins.

Acute toxicity study

Results of the acute toxicity study showed that there was no mortality or any significant change in the behaviour of mice recorded up to 5000 mg/kg of the plant extract. Based on the results of the preliminary toxicity test, the doses for further studies were decided to be 100, 200, and 400 mg/kg body weight of the rats as shown in Table 2.

Effect of the ME of E.guineensis (ME) on egg albumin-induced paw oedema in rats

Table 3 of the ME of E.guinneensis showed that 200 mg/kg significantly at P < .0001 elicited a dose-dependent activity with increasing degrees of inhibition of inflammation all through the duration of the experiment. The dose level of 100 mg/kg only showed little significant anti-inflammatory activity at the fifth and sixth hours, whereas 400 mg/kg showed no inhibition. The oedema inhibition was found to be comparable to that obtained for indomethacin (10 mg /kg).

Effect of solvent–soluble fractions of E. guineensis ME 200 mg/kg on egg albumin-induced rat paw oedema

Table 4 shows the anti-inflammatory activities of the extract and solvent–soluble fractions were compared at the most active dose level of 200 mg/kg, they were all found to have significant inhibitory activity all through the experimental period, with the fractions showing much higher activities than the crude extract.

Effect of ME of E. guineensis on formaldehyde-induced arthritis paw oedema in rats.

Table 5 shows the effect of the ME of E. guineensis on formaldehyde-induced arthritis paw oedema in rats. The results show that all dose levels of the crude extract resulted in significant inhibitory activity all through the duration of the experiment, except the 100 mg/kg dose level which showed the least anti-inflammatory activity, while the 200 mg/kg dose level showed the best activity profile, with the highest degrees of inhibition (for the extract) at both Days 1 and 10 and a more consistent activity in the 50s and 40s across the experimental period, with the 400 mg/kg dose having intermediate activity.

Effect of solvent–soluble fractions of E. guineensis ME 200 mg/kg on formaldehyde-induced arthritis paw oedema in rats

Table 6 shows the results of the extract and solvent–soluble fractions that were compared at the most active dose level of 200 mg/kg, it is observed that, while they all showed a similar V-shaped activity pattern as above, although with peak activities at the end of the experiment rather than the beginning, only the HF showed significant inhibitory activity all through that period. Also, although their activities peaked at higher inhibition levels than for the crude extract on Day 10, the other fractions had more instances of very low inhibition than the crude and also showed instances of ‘no inhibition’ on Days 3 and 4, suggesting less consistent activity than the crude extract-quite unlike in the previous model.

Discussion

The preliminary phytochemical investigations were studied to explore the bioactive phytochemical screening of the crude extract and its fractions present in the plants. The study reveals the presence of alkaloids, glycosides, flavonoids, tannins, steroids, and saponins crude extract of E. guineensis, with saponins being the most prevalent phytoconstituent and glycosides the least. But out of all the fractions, the MF had more alkaloids and flavonoids than saponins or steroids, while the HF had more steroids and resins than glycosides. However, the EF contained no steroids and very modest levels of tannins, alkaloids, flavonoids, resins, and saponins. (Table 1).

Methanol leaf extract and fractions of E. guineensis were used to evaluate the egg albumin-induced hind paw oedema test and formaldehyde-induced arthritis. Injection of egg albumin into the sub-planar surface of the right hind paw of mice induced oedema, which was measured as a function of the dose and time of administration of the products. The egg albumin model of inflammation induction is based on the generation of oedema by allergenic proteins contained in egg white [10], with major allergens like ovomucoid, ovalbumin, ovotransferrin, and lysozyme having been identified in that regard [11]. Leukocyte migration has also been primarily associated with the acute oedematous response to egg albumin injection and has been postulated to be mediated by histamine- and serotonin-induced increase in vascular permeability [12]. However, other mechanisms have been postulated to account for more prolonged egg albumin-induced inflammation lasting up to 5–6 h post induction [13] (Table 3). As the ME and fractions of E. guineensis demonstrated anti-inflammatory activities from the first hour up until the sixth, it is very likely that more than one pharmacological mechanism is possibly involved.

The comparative relationship observed for the ME and its fractions based on the degree and consistency of their inhibitory activities indicates that the n-hexane fraction, HF, showed the greatest anti-inflammatory activity while the methanol extract (ME) showed the least, with the activities of the EF and MF being intermediate in that order. Various anti-inflammatory studies have also found the n-HF to have the most potent activity [14–16]—thus, this finding is not strange, as the activity observed decreased with increasing polarity of the solvent used for fractionation, it suggests that the substance implicated in the activity is very likely non-polar. Based on the ‘like dissolves like’ principle [17], nonpolar substances tend to dissolve in nonpolar solvents, like n-hexane, and are sparingly soluble in water and other highly polar solvents, like methanol [18] As such, the phyto-constituent responsible for this activity should be most abundant in the HF and also decrease in abundance as the polarity of the fraction increases. While the HF showed an abundance of resins and steroids, a detailed scrutiny of the results of phytochemical analysis in Table 1 implicates resins as the most likely phyto-constituent involved in this activity.

Natural plant resins have been associated with many biological and medicinal activities, including anti-inflammatory activity [19] with their triterpenoid constituents being most implicated in this activity [20]. The anti-inflammatory activity of the frankincense resins has been found to be due to the pentacyclic triterpenoic acids, α- and β-boswellic acids and their derivatives [21, 22], which act by blocking the synthesis of leukotrienes and prostaglandins through 5-lipoxygenase enzyme and microsomal prostaglandin E2 synthase-1 inhibition, respectively [23, 24]. Another triterpenoid, mansumbinoic acid, from Commiphora mukul, has been reported to have potent anti-inflammatory activity in the rat paw oedema model [25, 26] as also has been reported for α, β-amyrone, a pentacyclic triterpene isolated from Protium paniculatum oilresins [27] It is possible that a triterpenoid may also be involved in the anti-inflammatory activity of the HF in the present study, and probably acting through similar pharmacological mechanisms as reported, but further studies are needed to confirm this.

Formaldehyde-induced arthritis is considered an important in vivo antiarthritic model because of its resemblance to human arthritis [28]. Following sub-plantar injection, formaldehyde induces arthritis in two phases: a primary direct paw engorgement phase attributable to local release of histamine, serotonin, and kinin-like substances and a latter more painful, aggravated inflammatory phase due to central release of prostaglandin-like substances [29, 30]. The wave-like inflammatory response seen in the negative control group in our experiment is descriptive of this biphasic response, and therefore serves to validate our experiment. However, the V-shaped activity pattern described in the discussion section for both the extract and fractions and the fact that they all showed much higher % inhibition at the latter part of the experiment than the earlier part suggest that the extract and fractions have greater effect on the latter centrally mediated phase than on the initial local phase of formaldehyde activity. Based on Table 6, this observation was much more acute for the methanol and EFs than for the crude extract and the n-HF, with the former showing little or no inhibition in the earlier phase. These observations suggest pharmacological differences in the fractions that require further studies to confirm.

The comparative relationship between the crude extract and its fractions in this model shows that the HF yielded the greatest anti-arthritic activity, just as in the previous model. However, unlike for the previous model, the crude extract showed an initially higher and generally more consistent activity than the other fractions, although with much less activity by the end of the experiment. Based on the relative polarities of the solvents and the ‘like dissolves like’ principle described in the previous section, it is plausible that at least two active phyto-constituent of contrasting polarities are involved in this anti-arthritic activity. In this hypothesis, the highly non-polar constituent(s) would account for the activity of the HF, while the polar one(s) would account for the activities of the MF and EF. As herbal extracts based on these solvents have been associated with anti-arthritic activity [31], this hypothesis is not unfounded. While the hypothesis would need to be validated in further studies, a detailed scrutiny of the results of the phytochemical constituents of the extract and fractions in Table 1 would suggest the involvement of a mix of some non-polar steroids and some relatively polar alkaloids and/or flavonoids in the activity. This hypothesis is buttressed by reports from previous studies which have highlighted the involvement of a mix of these phyto-constituents in anti-arthritic activity [32–34]

Plant-based steroids—also called phytosterols—have been associated with many medicinal effects, including anti-inflammatory activity [35] with their clinical efficacy being highly dependent on their lipo-solubility [36]—which supports the hypothesis above.

Various mechanisms have been reported for their anti-inflammatory actions—including inhibiting macrophage—and neutrophil-mediated inflammatory process; reducing oedema and proinflammatory cytokine levels and interfering with matrix degradation mediators, among others [37–40]

Stigmasterol, a very common unsaturated tetracyclic triterpene phytosterol in vegetable fats or oils from various plants, expresses its anti-arthritic activity via the mTORC1 signalling pathway, and improves cellular healing and joint health by deactivating certain inflammatory mediators [41, 42].The sterols implicated in the current study could act through any of these mechanisms.

Various reviews have shown that many of the alkaloids with medicinal activity show anti-inflammatory property. In Barbosa-Filho et al.’s review [42] of the 171 alkaloids evaluated between 1907 and 2000, they found 137 (80.1%) to have anti-inflammatory actions, with the isoquinoline derivatives being the most studied. A similar outcome was reported by [43], whose review of naturally sourced alkaloids with anti-inflammatory effects reported from 2000 to 2010 found that 40 of the 49 substances reported in the NAPRALERT data bank demonstrated anti-inflammatory activity, with the isoquinolines also being prominent among them. [44] identified berberine, tetrandrine, and stephanine as the most commonly occurring isoquinolines among Chinese medicinal plants, and their mechanism of action to be by inhibiting increased release prostaglandin E2 (PGE2) in in formaldehyde-induced arthritis. Other mechanisms associated with the anti-inflammatory activities of alkaloids include inhibiting neutrophil and monocyte functions [45] inhibiting cyclooxygenase (COX)-2 and nitric oxide synthase (iNOS) activity [46] and inhibiting the expression of TNF-α, and IL-6 [47] These mechanisms could also account for the anti-arthritic activity of any alkaloids in our extract.

Similar mechanisms as above have been described for the naturally occurring flavonoids (or bioflavonoids) that have demonstrated anti-inflammatory activity [48]. While a large range of flavonoids have been studied in inflammation, quercetin (and its derivatives), which is among the most common flavonoid in human diet, has a prominent place [28]. However, while Quercetin has been reported to be effective in formaldehyde-induced arthritis, it appears to be more effective against the inflammatory pain associated with the second phase of formaldehyde’s activity. This finding is therefore in agreement with our hypothesis based on our results.

Conclusion

We conclude that the current study has further validated the ethnomedicinal anti-inflammatory use of E. guineensis in inflammatory conditions. This study adds to previous knowledge in this area by highlighting the herb’s activity in sub-acute/chronic inflammation and arthritis. Also, the phytoconstituents likely involved in the pharmacological activities were highlighted. However, further studies are required to elucidate the specific pharmacological mechanisms involved.

Acknowledgement

We wish to thank Pharm Akahchukwu and, Pharm Peter Ikechukwu, of the department of pharmacology and toxicology for their help in overseeing the experiment. This work was part of my M.Sc. thesis and was not funded by any group. The author wishes to acknowledge the cares and guidance, he received during the experiment leading to unrestricted access in the course of the work. The author would also like to thank the anonymous reviewers for their input and suggestion during the review process.

Conflict of interest

None declared.

Data Availability

Data underlying this article are available in the article and online supplementary material.

References