Noni (Morinda citrifolia L.) fruit juice reduces paw edema and protects gastric mucosal injury in rats

Inflammation is a defense mechanism of the tissue against pathogens, damaged cells, toxic compounds, or irradiation [1]. However, chronic inflammation may cause tissue damage leading to the death of healthy cells [2]. The inflammatory reaction is caused by releasing various inflammatory mediators and is easily recognized as five cardinal signs such as fever, pain, redness, edema, and loss of function [3, 4]. A previous study mentioned that edema was reliable for evaluating the anti-inflammatory process [5].

The inflammatory process includes the regulatory role of prostaglandins and the primary cytokine mediators including cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) [6]. The most frequently given anti-inflammatory drugs for both acute and chronic illnesses are non-steroidal anti-inflammatory drugs (NSAIDs) [7]. Unfortunately, NSAIDs altered the surface of the gastric mucosal layer, causing back diffusion of hydrogen ions, and pepsin resulted in cell damage. Consumption of NSAIDs increases aggressive factors and decreases defensive factors that can develop gastric ulcers [8].

Noni (Morinda citrifolia L.) fruit is safe to be consumed for an extended period and does not affect the gastric mucous [9]. Noni fruit is known for its bioactive compounds, including scopoletin, alizarin, rutin, asperulosidic acid, nonisade A, and tricetin which can potentially decrease inflammation [10, 11]. This decreased inflammation might be caused by inhibiting COX-2, reducing the expression of nitric oxide, reducing the expression of IκB kinase (IKK) α/β, an inhibitor of kappa B alpha (I-κBα), and nuclear factor kappa B (NF-κB) p65 in lipopolysaccharide (LPS)-stimulated macrophages [10]. Noni fruit has a gastroprotective effect because of its polyphenol and metabolite content [12]. This research aimed to assess the effect of noni fruit as an anti-inflammatory agent in rat paw edema and gastric mucosal injury.

Animal study

Twenty male rats, aged 2-3 months and weighing 150-200 g, underwent 7 days of adaptation. Rats were kept in an environment with a consistent temperature and humidity level, a twelve-hour light/dark cycle with ad libitum access to food and water. The Medical and Health Research Ethics Commission, Faculty of Medicine, Diponegoro University, provided its ethical permission (No. 66/EC/H/FK-RSDK/VI/2018).

Noni juice preparation

The fruits were collected from Jepara, Indonesia, and brought to the Animal Laboratory of Diponegoro University, Indonesia. The seeds and skin at 390 g of noni fruits were removed before processing. 130 g of noni juice was centrifuged at 3000 rpm for 20 min until the supernatant was clear. The supernatant (60 cc) was used at 100% and 90% concentrations (diluted with physiologic saline). The juice was given at 1 cc orally right after the carrageenan injection.

Carrageenan-induced paw edema

The rats were divided into five groups such as control, carrageenan only, carrageenan and diclofenac sodium, carrageenan and 90% noni, and carrageenan + 100% noni. Carrageenan (Gumindo Perkasa Industry, Indonesia) was used to induce paw edema in rats. 30μl of 1% carrageenan (diluted in distilled water) was injected into four groups except the control group as previously described [5]. A plethysmometer was used to measure paw edema volume (ml) for  0, 15, 30, and 60 min post-injection. We defined paw edema volume changed as Δn (n = 1-4; 1 = reducing paw edema volume from 0 to 15 min; 2 = reducing paw edema volume from 15 to 30 min; 3 = reducing paw edema volume from 30 to 60 min; and 4 = reducing paw edema volume from 0 to 60 min).

Gastric mucosa examination

Animals were terminated, and the stomach was removed for further examination. For 24 hours, the stomach was fixed in 10% buffered. Gastric mucosal integrity was examined through a histopathological examination using hematoxylin-eosin (HE) staining and the severity of mucosal injury was assessed using the Barthel Manja score [3]. We used an Olympus CX23 microscope (Olympus, Japan) for histopathological examination of the gastric mucosa.

Statistical analysis

The paw edema volume showed normal distribution using the Saphiro-Wilk method except in the carrageenan + 100% noni group. Repeated ANOVA was used to compare paw edema volume in 0-, 15-, 30-, and 60-minute post-carrageenan injection in control, carrageenan + diclofenac sodium, and carrageenan + 90% noni groups, while the carrageenan + 100% noni group were analyzed using the Friedman test. The one-way ANOVA was used to assess Δ2 and Δ4 to find differences across groups and a post hoc test was used to find differences between groups. The Kruskall-Wallis and Mann-Whitney test was used to examine gastric mucosa’s Δ1, Δ3, and histopathological features. The results were presented as the mean ± SD. When, p< 0.05, the differences were deemed significant. The SPSS software application was used to conduct statistical analysis.

All groups were observed for their behavior during the research. Indicators such as activity, the excess daily volume of water, and viability were observed. All those indicators were assessed semi-quantitatively. All groups were at the same activity levels and survived until the experiment was completed.

Effect of noni fruit juice on paw edema volume in rats

The results showed decreasing volume edema from 0 to 60 min after treatment in the carrageenan + diclofenac sodium group, carrageenan + 90% noni group, and carrageenan +100% noni group  (Figure 1). Paw edema volume in the group carrageenan was only adjusted based on the previous study [13]. The largest paw edema volume after being induced by 30 μl sub–plantar 1% carrageenan (0 min) was shown in the carrageenan + 90% noni group (1.30 ± 0.23 cc). At the last minute (60 min), the smallest paw edema volume occurred in the carrageenan + diclofenac sodium group (0.65 ± 0.23 cc). All groups showed a decrease in paw edema volume over time. We used repeated ANOVA and the Friedman test to assess these results, and the results revealed a significant difference (p< 0.05). These results proved that both groups with diclofenac sodium and noni juice administration have the potential to be anti-inflammatory with a decrease in paw edema volume.

Effect of noni fruit juice on paw edema volume in rats

The previous results showed differences in paw edema volume in the first minute (0 min) after carrageenan injection, so further analysis was needed as a comparison among groups. We analyzed the comparison with Δn. Δn was used as a more accurate parameter in comparing the effectiveness of reducing paw edema volume among groups. Figure 2 compares decreasing paw edema volume from 0 to 60 min. We performed Δn outcome analysis to compare among groups, the Kruskall-Walli’s test and Mann-Whitney test were used. Based on the analysis, almost all comparisons among groups showed no significant differences except for Δ3, the comparison between carrageenan + diclofenac sodium group and carrageenan + 90% noni group (p = 0.005). These results indicated no significant difference in the administration of diclofenac sodium with noni juice in reducing paw edema volume.

Effect of noni fruit juice on gastric mucosa integrity

The results of gastric mucosa integrity after being treated with noni fruit juice and diclofenac sodium can be seen in Table 1 and Figure 3.

The control group showed no or less pathological findings on the gastric mucosa (score 0 to 1). The carrageenan only group was not evaluated for the gastric mucosa histopathology. Based on Szandruk-Bender et al, administration of carrageenan only does not damage the gastric mucosa (score 0 = no damage) [14].  The carrageenan + diclofenac sodium group showed injury at the muscular level (score 2 to 3). The carrageenan + diclofenac sodium group score was higher than the carrageenan + 90% noni or carrageenan + 100% noni groups. The results of the carrageenan + 90% noni and carrageenan + 100% noni groups showed epithelial desquamation (score 1 to 2). The comparisons of all groups revealed significant differences according to the Kruskal-Walli’s test (p=0.002). Additionally, the Mann-Whitney test was used to determine the differences between groups. Except for the carrageenan + 90% noni and carrageenan + 100% noni groups, which did not demonstrate significant differences with p>0.05, the Mann-Whitney test revealed significant differences between groups with p<0.05. These results could prove that the effect of noni juice administration as an anti-inflammatory agent in rat’s paw edema was safer on the gastric mucosa integrity than diclofenac sodium administration.

Noni is famous for its chemical compounds that have various health benefits. Besides its nutrition, noni contains antioxidant, antimicrobial, anticancer, and anti-inflammatory properties [11]. Noni fruit contains scopoletin, alizarin, rutin, asperulosidic acid, nonisade A, and tricetin. The bioactive compound of noni fruit exhibited potential anti-inflammatory activity [10, 11]. Another study showed that RAW264 cells activated by LPS showed anti-inflammatory effects from Morinda citrifolia [15]. The anti-inflammatory effect was also demonstrated in extensive studies regarding the biological activity reported in Noni fruit juice, showing the strongest nuclear erythroid-related factor 2 (Nrf2) activation and NF-κB inhibitory activity [16]. This evidence is consistent with our results, which demonstrated a significant decrease in paw edema, an inflammation parameter in our study. Edema is a form of inflammatory response [3, 4]. Therefore, our study used carrageenan induction to produce edema as a form of inflammation. It was demonstrated in this study that administering noni juice reduced edema as a form of anti-inflammatory. These results also followed a previous study proving that topical noni leaf extract could reduce rat paw edema induced by carrageenan [17].

Noni fruit containing rutin, asperulosidic acid, nonisade A, and tricetin may have therapeutic and preventative effects on inflammatory diseases. In anti-inflammatory tests, the active compounds in noni fruit prevented LPS-stimulated macrophages from producing nitric oxide (NO), a pro-inflammatory mediator. Furthermore, other mechanisms of anti-inflammatory action include down-regulating the expression of IKKα/β, I-κBα, NF-κB p65 in LPS-stimulated macrophages, and down-regulating the expression of nitric oxide synthase and COX-2 [10]. More than that, antioxidants help to prevent inflammatory responses. Noni juice ameliorated oxidative stress by up-regulating the expression of related antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) and attenuating reactive oxygen species (ROS) and malondialdehyde (MDA) [18]. Manavalan et al. demonstrated the antioxidant potential of Noni fruit juice [16]. Scopoletin in noni fruit can reduce oxidative stress by increasing Nrf2 activation and inhibiting NF-kB [16, 19].

The difference in the reduction of paw edema defined as Δn between noni juice and diclofenac sodium groups was insignificant. It can be assumed that the potential anti-inflammatory effect between noni juice and diclofenac sodium is not much different. Carrageenan sub–plantar injection promotes histamine release, serotonin, prostaglandin, and bradykinin, whereas, after 1-hour injection, neutrophil infiltration, free radicals produced by neutrophils, NO, and pro-inflammatory cytokines such as tumor necrosis factor (TNF-α) were released [20]. According to reports, noni juice decreased carrageenan-induced paw edema by preventing COX-2 activity, preventing NO generation, and triggering prostaglandin E [10]. This mechanism is not too different from diclofenacsodium; it is through the inhibition of COX-1 and COX-2 relatively equally [21].

We examined the safety of using noni juice on the stomach because the anti-inflammatory side effects often cause gastric ulcers [22]. Our study showed that the noni juice group did not cause gastric mucosal injury. These results were significantly different from the group given diclofenac sodium. Although both anti-inflammatory mechanisms are not too other, the effect of noni juice is more selective as a COX-2 inhibitor. A previous study proved that Noni fruit acts as a COX-2 inhibitor [10]. Acute mucosal injury caused by diclofenac sodium as NSAID agent was commonly found and reversible [23]. The damage caused by selective COX-2 inhibitors was possible, even infrequent, depending on the subject's condition. Changes in gastrointestinal bacteria and free radicals increase the aggressive effect on the stomach, leading to mucosa injury [24]. Oxidative stress includes ROS, reactive oxygen intermediates (ROI), and the byproduct of cellular metabolism, which causes oxidative damage to the tissue. NSAIDs were one of the exogenous factors that caused ROS production [25]. ROS production induced lipid peroxidation on the epithelial cells of the stomach, and pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-1β (IL-1β) resulted in mucosa injury. The noni antioxidant supports the stomach defensive factor by compensating for ROS-induced epithelial damage and protecting further damage from oxidative stress [16, 19, 26]. Recent meta-analysis studies showed 19% selective COX-2 increased upper gastrointestinal side effects but less gastrointestinal toxicity compared with non-selective NSAIDs [27]. This was in accordance with our results, which showed that several samples using noni juice fruit scored 1-2 of the Barthel Manja score (epithelial desquamation) but less than a score when giving diclofenac sodium (score 2-3).

The anti-inflammatory effect in pure noni juice significantly decreased carrageenan-induced paw edema [28]. Yet, this study's 90% concentration of noni juice also significantly reduced paw edema volume. The study’s results, the comparison between both in terms of lowering paw edema and the histopathological appearance of the stomach was not significantly different. We can conclude that the 100% and 90% concentrations of noni juice have the same potency. However, more investigation is required to establish the optimal noni juice concentration through studies at lower concentrations.

This study only observes the acute effect of noni juice administration on gastric mucosa compared with diclofenac sodium. Noni juice was administrated instead of noni extract in this study. For further research, the author suggested administering noni juice extract instead of juice and observing this treatment's chronic effect on mucosa. This study also has other limitations in showing anti-inflammatory effects; further research is needed to measure pro-inflammatory markers.

The administration of noni juice could reduce paw edema as an anti-inflammatory effect. The anti-inflammatory effect between noni juice and diclofenac sodium had the same potential. The safety effect of noni juice was shown in the histopathological future of the stomach, which proved that noni juice could protect against gastric mucosal injury. Mucosa injury caused by noni juice treatment was not worse than diclofenac sodium because of the selective COX-2 and the antioxidant activity of quercetin and scopoletin. Otherwise, COX-2 inhibitors in diclofenac sodium cause mucosal injury due to oxidative stress.

None.

All authors developed the ideas, designed the study, conceptualized, collected data, analyzed the data, drafted the manuscript, and revised and edited the manuscript; DA supervised the study. The final manuscript has been read and approved by all authors.

There is no conflict of interest among the authors.