Assessment of the anti-inflammatory and analgesic effects of Opuntia ficus indica L. Cladodes extract

ABSTRACT

This study aimed to evaluate the anti-inflammatory and analgesic properties of the methanolic extract of Opuntia ficus indica L. in small animal (rats and mice model). The current treatment for febrile conditions often involves the use of non-steroidal anti-inflammatory drugs (NSAIDs), which can have adverse effects, particularly gastrointestinal ulcers. Therefore, there is a growing need to explore natural alternatives with fewer side effects. The study utilized various experimental models to assess the effects of the extract. The results demonstrated a significant analgesic effect of the extract, as evidenced by a reduction in pain induced by acetic acid and hot plate tests. Additionally, the extract exhibited anti-inflammatory effects, as indicated by a decrease in carrageenan-induced paw edema and dextran-induced inflammation. To gain insights into the chemical composition of the extract, HPLC analysis was conducted. The analysis successfully identified and quantified 20 compounds, including luteolin, galangin, catechin, thymol, methylated quercetin, quercetin, rutin, acacetin, hesperidin, apigenin, kaempferol, pinocembrin, chrysin, gallic acid, caffeic acid, ascorbic acid, ferulic acid, m-coumaric acid, rosmarinic acid, and trans-cinnamic acid.The findings suggest that Opuntia ficus indica L. extract holds promise as an effective and reasonably priced natural remedy for pain and inflammation in rats and mice model. The comprehensive chemical composition analysis provided valuable insights into the presence of various bioactive compounds, which may contribute to the observed therapeutic effects. Further research and exploration of the extract’s mechanisms of action are warranted to fully understand its potential in small animal healthcare.

KEYWORDS:

• Anti-inflammatory
• analgesic
• HPLC
• opuntia ficus indica L. extract
• medicinal plants

1. Introduction

Human traditions have developed the knowledge and use of medicinal plants to improve human health [1]. The medicinal properties of plants are due to products synthesized by the plants themselves called secondary metabolites Secondary metabolites, particularly polyphenols, serve as antibiotics in a comprehensive sense, as they play a crucial role in safeguarding plants against a variety of threats including fungi, bacteria, animals, and even other plants [2]. These compounds, distinct from primary metabolites that are essential for basic cellular functions, are synthesized by plants as a means of defense against potential pathogens and predators. Polyphenols, among other secondary metabolites, exhibit antimicrobial properties, effectively inhibiting the growth and proliferation of various microorganisms. Through their antibiotic activities, these secondary metabolites contribute significantly to the overall resilience and survival of plants in their natural ecosystems. Algeria, given its privileged biogeographic position and its extension between the Mediterranean and sub-Saharan Africa, is considered among the countries known for their floristic diversity [3], to which is added a secular tradition of traditional use of plants. There are about 3000 species of plants, of which 15% are endemic. The potential of medicinal plants encompasses a vast array of species, presenting diverse attributes that have garnered significant attention in the realm of scientific investigation [4]. The plant Opuntia ficus-indica, commonly referred to as prickly pear, is a member of the Cactaceae family. The Cactaceae family is known to encompass approximately 130 genera and nearly 1500 species, originally indigenous to the New World [5]. The distribution of the prickly cactus pear (Opuntia ficus-indica) extends across Mexico, a substantial portion of Latin America, South Africa, and the Mediterranean region [6]. Opuntia genus plants have found various applications, encompassing (1) the extraction of carminic acid, (2) culinary purposes, (3) utilization for their functional attributes, and (4) other diverse applications. Concerning the extraction of carminic acid, this compound is obtained through a process involving milling and desiccation of Opuntia plants, followed by the extraction from the female cochineal insect, which thrives on the succulent branches of these plants [7]. In this specific context, our focus centers on the comprehensive examination of Opuntia ficus indica L., a plant species of particular interest. Opuntia ficus indica harbors bioactive compounds known as betacyanins, which manifest as reddish pigments and possess noteworthy antioxidant and anticancer properties. Consequently, these bioactive molecules can be harnessed for their therapeutic potential in natural treatments. Notably, both the flowers and extracts derived from Opuntia ficus indica demonstrate remarkable anti-inflammatory effects on the human body [8].

2. Materials & methods

2.1. Plant material and extraction

The cladodes of Opuntia ficus indica L. were collected in the wilaya of Elbayadh (Algeria).

Figure 1. The region where cladodes of Opuntia ficus indica L. were collected, ElBayadh, Algeria; data source: own illustration.

The plant material that was collected underwent a drying process by placing it on paper at room temperature and exposing it to sunlight for a duration of 7 days (figure 1). The aerial part of the plant was air dried and subsequently pulverized into a fine powder. A total of 500 grams of this powder were subjected to a cold maceration process in 2.5 liters of methanol for a period of 72 hours, with regular shaking to enhance extraction efficiency. After this maceration period, the solution was separated by decantation, and the resulting extract was filtered to remove any impurities. The filtrate was then subjected to evaporation at 40 degrees Celsius until complete dryness was achieved, resulting in the formation of a concentrated extract.These experimental procedures were employed to extract and concentrate the bioactive constituents present in the plant material, enabling subsequent analysis and evaluation of its chemical composition and potential biological activities.

2.2. Experimental animals

Sixteen male Wistar albino rats, weighing between 150–200 grams, and sixteen Swiss mice, weighing between 20–30 grams, were procured from the Pasteur Institute in Algeria. The animals were individually housed in plastic cages under controlled conditions. The temperature in the animal facility was maintained at 23 ± 2 degrees Celsius, with a relative humidity range of 50–55%. The animals were exposed to a 12-hour light/12-hour dark cycle, both before and during the experimental period.

Throughout the study, the animals had unrestricted access to standard laboratory chow and water, provided ad libitum. The animals’ diets were not restricted in any way during the course of the experiments.

All animal handling procedures and experimental protocols were conducted in strict adherence to the established standards and guidelines. The Animal Ethics Committee of the Department of Biology at the University of Saida approved the experimental protocols employed in this study, ensuring the ethical treatment and welfare of the animals throughout the research process.

2.3. Anti-inflammatory activity

2.3.1. Carrageenan-induced paw edema

For this particular study, a total of four groups, each consisting of four Wistar rats weighing between 150–200 grams, were utilized. The experimental groups were administered the extract orally at a dose of 40 mg/kg. In the positive control group, an oral administration of 10 mg/kg indomethacin was performed, while the negative control group received an oral administration of distilled water at a volume of 4 mL/kg.

After one hour of administration, a 0.1 mL injection of a 1% w/v carrageenan suspension in 0.9% isotonic saline was injected into the subplantar tissue of the right hind paw of each animal. The thickness of the paw was measured using a Vernier caliper at two time points: initially at 0 hour (baseline measurement) and subsequently at a 1-hour interval for a total of 5 hours [9]. These experimental procedures were conducted to evaluate the potential anti-inflammatory effect of the administered extract by assessing changes in paw thickness as an indicator of inflammation. The positive control group (indomethacin) and the negative control group (distilled water) serve as reference points for comparison to the test groups, enabling the assessment of the extract’s efficacy in reducing inflammation.

2.3.2. Dextran-induced paw oedema

Wistar rats weighing between 150 and 200 g were randomly divided into four groups of four animals each for this study. The test group received the extract orally, while the negative control group received distilled water (4 ml/kg) orally. The reference group received 60 mg/kg diphenhydramine. The animals were treated one hour before receiving an injection of 0.1 ml of 1.5% w/v dextran in 0.9% isotonic solution into the subplantar tissue of their right hind paw (7 [10]. Calipers were used to measure paw thickness at 0, 1, 2, 3, 4 and 5 hours [11].

2.3.3. Xylene-induced ear oedema

For this study, four groups of four male Swiss albino mice were used. The extract was administered orally to the test groups. The method of [12] was adopted for this study. Briefly, “distilled water (4 mL/kg) was administered to the negative control group while the reference group received dexamethasone (1 mg/kg). After 30 minutes, oedema was induced in each group of mice by applying a drop of xylene to the inner surface of the right ear. Approximately 15 minutes later, the animals were sacrificed and both ears were cut, sized and the weight was taken and recorded.

2.4. Analgesic activity

2.4.1. Twist test

Acetic acid is the most widely used chemical agent for assessing the peripheral analgesic activity of medicinal plants. Intraperitoneal (IP) injection of acetic acid causes inflammatory pain by inducing capillary permeability [13] and induces stereotypic behaviour in rats characterised by abdominal contractions. However, although the acetic acid test is widely used to test the analgesic effect.

Five minutes after injection of acetic acid, painful syndromes in the rats tested appeared, characterised by stretching movements of the hind legs and twisting of the dorso-abdominal musculature.

Four groups of four male Swiss albino mice were used in this study. The test groups received the extract (40 mg/kg) orally, while distilled water (10 ml/kg) was administered to the untreated induced group (negative control). The reference drug used was aspirin (100 mg/kg). Thirty (30) minutes after the administration of the reference drug and the extract, acetic acid in normal saline (0.6% v/v) was administered to the mice by intraperitoneal injection and the torsions were counted for 30 minutes at five-minute intervals [14].

2.4.2. Formalin test

In this study, male Wistar rats were randomly divided into four groups, with four animals in each group. The test group received an oral administration of the extract at a dose of 40 mg/kg, while the reference group received subcutaneous administration of aspirin at a dose of 100 mg/kg. The negative control group received an oral administration of distilled water.

After a time interval of 30 minutes from administration, a subcutaneous injection of 20 µL of 1% formalin was administered into the right hind leg of each rat. The pain response was assessed by measuring the time spent biting and licking the injected paw, recorded in seconds. The observation of pain-related behaviors was conducted for a duration of 5 minutes following the formalin injection [15].

This experimental setup was employed to evaluate the analgesic activity of the administered extract by assessing its effect on pain response in the formalin-induced pain model. The reference group, treated with aspirin, served as a comparative standard, while the negative control group allowed for the evaluation of any inherent pain response in the absence of treatment.

The measurement of pain-related behaviors provides valuable insights into the potential analgesic properties of the administered extract, allowing for a comparison of its efficacy with the reference drug. The results obtained from this study contribute to the understanding of the extract’s potential as a pain-relieving agent and its potential application in pain management.

2.4.3. Hot plate test

The hot plate test is one of the most widely used tests of nociception based on a high-intensity stimulus. The pain induced by the thermal stimulus must pass through the central nervous system (CNS) [16].

The jumps observed in this test involve a voluntary motor act and is considered to be unlearned, and sustained by the activation of supraspinal sensory nerve circuits of very complex organisation [17].

Our Swiss mice were divided into four groups of four mice each. The animals were individually placed on a hot plate maintained at a constant temperature of 50°C, the time interval between placement and the paw shake or lick or jump was recorded as an index of response latency. The latency period before the drug was determined and recorded for each animal. The negative control group was treated orally with distilled water at 10 ml/kg. The test animal groups received the extract (40 mg/kg). Pentazocine (15 mg/kg) was administered intraperitoneally and used as standard. The animals were placed on the hot plate at 15, 30, 45, 60 minutes, 15 minutes after treatment and the time for paw stirring or jumping was recorded.

2.5. Statistical analysis

The data from this study were expressed as mean ±SEM. The means of the different groups were compared by ANOVA. P values < 0.05 (95% confidence interval) were considered significant.

3. Results

3.1. Carrageenan-induced paw edema

At 40 mg/kg, the Opuntia ficus indica L extract considerably reduced paw oedema in comparison to the negative control group. As comparison to indomethacin, the 30 mg/kg extract showed a higher degree of paw oedema inhibition within the first hour. The 40 mg/kg extract, when compared to both groups, exhibited a non-significant impact at the fourth hour but demonstrated paw oedema inhibition in the fifth hour. The study examined at the Opuntia ficus indica L. methanolic extract’s ability to reduce inflammation and provide pain relief. Carrageenan proven to be an unrivaled choice as an agent for evaluating anti-inflammatory medications due to its non-antigenic character and the eradication of the subsequent systemic impact [18]. Carrageenan-induced oedema includes two stages, according to Riahi et al. Within an hour following carrageenan-induced inflammation, the body enters the first phase, which is brought on by mast cell production of cytoplasmic enzymes, serotonin, and histamine. Platelet activating factor and arachidonic acid metabolites each have specific functions to perform [19]. After an hour, carrageenan-induced oedema enters its second phase, which is mediated by the production of enzymes that break down proteins, prostaglandins, oxygen, free radicals, arachidonate metabolites, neutrophil migration, and other mediators released from neutrophils [20] and kinins are in charge of maintaining consistency across the two stages [21].Our results showed that the extract significantly (p < 0.05, 0.01) inhibited paw oedema by producing an inhibitory effect in the first and second phase of inflammation. The antihistaminic potential of the extract is demonstrated in the first phase, which may be the result of the ability of the extract to reduce carrageenan-induced leakage from the microvasculature [22]. Our study is in agreement with [23]. The authors of this study observed a reduction in oedema in albino rats treated with Opuntia ficus indica L seeds dissolved in petroleum oil extract. Due to the fact that the carrageenan-based inflammatory model displays prostaglandin activities, the potential for improvement of oedema in the second phase also supports a potential suppression of cyclooxygenase production [24].

3.2. Dextran-induced paw oedema

We observed that animals treated with our extract (15 and 30 mg/kg) reduced paw oedema compared to the negative control. Furthermore, dextran-induced paw oedema is thought to be mainly mediated by serotonin and released by mast cells [25]These released inflammatory mediators result in marked vascular changes such as vasodilation, increased permeability and slowed blood flow, ultimately leading to paw inflammation. Our study demonstrated that the ethanolic cladode extract of Opuntia ficus indica L significantly (p < 0.05, 0.01) inhibits dextran-induced paw oedema. These observations are in agreement with the study reported by [26] where they showed that ethanolic extract of Leptadenia arborea demonstrated an inhibitory effect on oedema size.

3.3. Xylene-induced ear oedema

Our study investigated the anti-inflammatory effects of our extract at a dose of 40 mg/kg using the xylene-induced ear edema model. This model is commonly employed to assess the efficacy of anti-inflammatory steroids and is considered less sensitive to non-steroidal anti-inflammatory agents [27].

The xylene-induced ear edema model provides indicators of acute inflammation, including inflammatory cell infiltration, severe vasodilation, and edematous changes in the skin. In our study, we observed that the administration of our extract inhibited xylene-induced ear edema compared to the negative control group. These findings are consistent with a previous study conducted by [28], where they demonstrated the inhibitory potential of Crinum glaucum in xylene-induced ear edema. Our results further support the notion that our extract possesses anti-inflammatory properties, as evidenced by the reduction in inflammatory edema in the xylene-induced model.

The inhibition of xylene-induced ear edema serves as an important indication of the anti-inflammatory activity of our extract. These findings contribute to the growing body of evidence supporting the therapeutic potential of natural compounds in the treatment of inflammatory conditions. Further research and exploration of the underlying mechanisms are warranted to fully understand the anti-inflammatory properties of our extract and its potential applications in the field of inflammation-related disorders.

3.4. Mouse torsion test

The nociceptive response was assessed by measuring the number of cramps produced by the animals following intraperitoneal injection of acetic acid. The table presented the recorded number of cramps, along with their standard deviation, and the percentage of cramp inhibition. The results obtained from this test demonstrated a significant reduction (p < 0.001) in the number of cramps induced by acetic acid when treated with the extracts of our studied plant. Notably, the observed effect was more pronounced compared to the reference drug (aspirin), indicating a greater antinociceptive activity of our extract. The maximum antinociceptive activity was achieved with our extract, further highlighting its potential as an effective analgesic agent.

Comparing our results with a previous study conducted by [29] on the extract of Hibiscus sabdariffa calyces, it was found that their extract, administered at a dosage of 400 mg/kg, inhibited the number of twists by only 7.8%. This percentage of inhibition is considerably lower than the results obtained in our study.These findings demonstrate the significant antinociceptive activity of our studied plant extract, surpassing the effects observed with the reference drug and exceeding the inhibitory effects reported in a comparable study. Further research and investigation are necessary to elucidate the underlying mechanisms of action and potential therapeutic applications of our extract in the field of pain management.

3.5. Formalin test

In the formalin-induced test, the extract improved formalin-induced pain compared to the negative control group. This observed reduction was seen in both phases. The 40 mg/kg dose had an effect in both phases similar to that of aspirin.

One of the most widely used methods for assessing anti-nociceptive activity is acetic acid-induced twisting of animal models [30]. This method is very sensitive, even at lower doses, compared to the tail-prick test for detecting the anti-nociceptive potential of bioactive agents [31]. Our study showed that the extract caused a significant reduction in acetic acid-induced biting at all doses. The extract was generally more effective than the 100 mg/kg dose of aspirin, which was the control drug used, and was maintained throughout the 30 minute period, suggesting peripheral mediation for the analgesic effect of the extract.

3.6. Hot plate test

When compared to the control group, the extract reduced the amount of time that mice were in discomfort after being placed on a hot plate (Table 4). Pentazocine (15 mg/kg), a medication recognized for its central activity, showed a stronger inhibiting impact than the groups who received extract treatment. Based on the fact that central analgesics like tramadol increase the pain threshold by inhibiting the formation of prostaglandins, the hot plate studies were conducted [32]. However, additional anti-nociceptive processes may have allowed the extract to operate. In fact, activating K±ATP channels that allow the buildup of intracellular Ca++, which in turn starts a cascade of secondary messengers, can prevent thermal hyperalgesia [33]. Our results revealed that the extract considerably increased the latency duration in the hot plate experiment, indicating that the central nervous system had a major role in mediating this analgesic effect. The analgesic effect reported in this study is considerable in compared to earlier studies [34] where rats treated with aqueous extract of Balbisia calycina at a concentration of 400 mg/kg did not affect the latency duration.

To evaluate centrally mediated nociceptive effects, hot plate experiments are frequently employed; In our study in Table 1,Table 2,Table 3, we observed that our extract was able to induce a significant extension of the pain response latency on the hot plate, as shown in Table 4, suggesting that the analgesic activity was centrally mediated. The abnormal calmness observed a few minutes after the administration of the extract in the animal models suggests psychotropic effects of the extract.

Table 1. Effect of methanoic extract of Opuntia ficus indica L cladodes on carrageenan-induced paw oedema.

Groups	Doses (mg/kg)	Pre-drug	0hr	1hr	2hr	3hr	4hr	5hr
Witness (-)	0.5	-	0.60±0.02	0.67±0.02	0.71±0.03	0.63±0.01	0.77±0.02	0.69±0.01
Indomethacin	10	0.36±0.02	0.49±0.04	0.51±0.03*	0.69±0.01	0.58±0.02	0.54±0.03*	0.51±0.02**
O.ficus indica	40	0.31±0.01	0.52±0.02	0.53±0.03**	0.66±0.03	0.60±0.04	0.59±0.03	0.58±0.02*

Values are mean ± SEM; n=4; *=p<0.05.

Table 2. Effect of methanolic extract of Opuntia ficus indica L on xylene-induced ear oedema in rats.

Groups	Doses (mg/kg)	Left ear (g)	Right ear (g)	Differences in both ear (g)
Witness (-)	0.5	0.011±0.00	0.018±0.00	0.007±0.00
Aspirin	100	0.062±0.01	0.087±0.02	0.025±0.02***
O.ficus.indica	40	0.061±0.01	0.104±0.0	0.039.01**

Values are mean ± SEM; n=4; **p<0.05.

Table 3. Effect of ethanolic extract of Opuntia ficus indica L cladodes on acetic acid-induced spasm as a function of time.

Groups	Doses (mg/kg)	0–5 mins	5–10 mins	10–15 mins	15–20 mins	20–25 mins	25–30 mins
Witness (-)	0.5	17.0±6.09	26.9±2.10	26.0±2.38	19.8±3.27	18.0±2.58	15.3±1.19
Aspirin	100	7.15±1.32*	19.0±1.00**	21.5±1.56	20.5±1.26	17.4±2.18	15.4±1.18
O.ficus.indic	30	0.0±0.0**	15.1±1.96***	15.6±2.16**	10.59±1.94**	8.58±2.18*	7.41±1.14***

Values are mean ± SEM; n=4; p<0.05.

Table 4. Effect of ethanolic cladode extract of Opuntia ficus indica L on hot plate induced pain as a function of time.

Groups	Doses (mg/kg)	0 sec	15 mins	30 mins	45 mins	60 mins
witness (-)	0.5	3.00±0.32	4.27±0.75	3.20±0.22	3.10±0.34	2.87±0.46
Pentazocine	100	2.15±0.13	6.75±2.00*	7.40±1.82**	10.92±0.89***	9.90±1.68***
O.ficus.ind	40	3.51±0.43	6.04±2.57	5.67±2.38*	8.09±1.79**	8.11±1.79**

Values are means ± SEM; n=4; p<0.05.

3.7. Determination of the chemical composition of an ethanolic extract of Opuntia ficus indica L. by HPLC

The chemical composition of the ethanolic extract of Opuntia ficus indica L. was analyzed using high-performance liquid chromatography (HPLC) techniques. The results of the analysis are summarized in Table 5.

Table 5. Composition of ethanolic extract of Opuntia ficus indica L. (EEA) by HPLC/UV (mg/g).

Peak Number	Compounds	Quantity (mg/g EEA)	Retention Time (min)
1	Luteolin	0.45	0.42
2	Galangin	0.11	0.59
3	Catechin	0.89	2.37
4	Thymol	0.12	2.68
5	Methylated quercetin	1.16	3.74
6	Quercetin	1.36	4.01
7	Rutin	0.26	4.57
8	Acacetin	0.17	5.25
9	Hesperidin	0.1	5.41
10	Apigenin	0.02	5.81
11	Kaempferol	0.30	6.73
12	Pinocembrin	0.19	7.18
13	Chrysin	0.95	7.252
14	Gallic acid	0.18	2.11
15	Caffeic acid	1.01	2.37
16	Ascorbic acid	0.12	2.68
17	Ferulic acid	1.24	3.74
18	m-Coumaric acid	0.33	4.01
19	Rosmarinic acid	0.41	4.57
20	Trans-cinnamic acid	0.11	6.73

A total of 20 compounds were successfully identified and quantified in the extract using HPLC. These compounds include luteolin, galangin, catechin, thymol, methylated quercetin, quercetin, rutin, acacetin, hesperidin, apigenin, kaempferol, pinocembrin, chrysin, gallic acid, caffeic acid, ascorbic acid, ferulic acid, m-coumaric acid, rosmarinic acid, and trans-cinnamic acid.

Among these compounds, quercetin (1.36 mg/g), ferulic acid (1.24 mg/g), methylated quercetin (1.16 mg/g), and caffeic acid (1.01 mg/g) were identified as the predominant constituents in the ethanolic extract. These compounds exhibited the highest concentrations among all the identified constituents.

The presence of these bioactive compounds, particularly quercetin, ferulic acid, methylated quercetin, and caffeic acid, in significant amounts indicates the potential pharmacological properties of the Opuntia ficus indica extract. These compounds have been associated with various beneficial effects, including antioxidant, anti-inflammatory, and anticancer activities, among others. The quantification of these compounds provides valuable information about the chemical profile of the extract, contributing to our understanding of its potential therapeutic applications.

4. Conclusions

Based on the traditional uses attributed to Opuntia ficus indica, our study aimed to comprehensively investigate its therapeutic properties and provide scientific validation for its anti-inflammatory and analgesic activity. The results of our study unequivocally demonstrate the strong anti-inflammatory activity of the plant extract in vitro, lending empirical support to its traditional usage as a natural remedy for inflammatory and painful conditions. These findings highlight the significant role of traditional medicinal practices in the treatment of diverse ailments and emphasize the potential of medicinal plants as valuable sources of active compounds for therapeutic purposes. The integration of traditional knowledge with scientific inquiry enhances our understanding of the medicinal potential of natural remedies and opens avenues for the development of novel therapeutic interventions.

Institutional Review Board Statement

All experiments complied with the Algerian legislation (Law Number 95–322/1995) inherent to protection of animals designed to experimental and other scientific purposes as well with the guidelines of the Algerian Association of Experimental Animal Sciences (AASEA) and were specifically approved by the latter (AASEA authorisation number 45/DGLPAG/DVA/SDA/14).

Acknowledgments

The authors extend their appreciation to Researchers Supporting Project number (RSP2023R390), King Saud University, Riyadh, Saudi Arabia and ‘Ministère de l’Enseignement Supérieur et de la Recherche Scientifique, Algeria’

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This Research was funded by le Ministère de l’Enseignement Supérieur et de la Recherche Scientifique, Algeria and the Researchers Supporting Project No. (RSP2023R390), King Saud University, Riyadh, Saudi Arabia.