INTRODUCTION:

Inflammation is a microcirculation-protective response that occurs as a result of infection or injury. Local and systemic inflammatory responses aim to remove the inciting stimulus, promote tissue repair and recovery, and establish immune memory in the situation of infection so that the host can mount a more specific and faster response to future contact¹. The important role of inflammation in various diseases such as cancer, atherosclerosis, reperfusion injury, diabetes, and Alzheimer's disease has driven major research efforts into processes that activate and control inflammatory responses^2,3.

Pro-inflammatory mediators are important regulators of physiological processes; however, uncontrolled production of pro-inflammatory mediators can maintain or enhance the inflammatory response, resulting in chronic inflammation. Cyclooxygenase exists in two isoforms: COX-1 and the inducible COX-2^4–6. Inhibiting these mediators can provide anti-inflammatory action. Nonsteroidal anti-inflammatory drugs (NSAIDs) or steroidal anti-inflammatory drugs (SAIDs) are used to inhibit the pathway of inflammatory mediators⁷. Nowadays, NSAIDs are some of the most widely prescribed medications in the world, and their analgesic, anti-inflammatory, and antipyretic properties are widely acknowledged. However, NSAIDs, like many other drugs, have a variety of adverse effects, such as cardiovascular (CV), renal toxicity, gastrointestinal (GI) events, high blood pressure, and worsening of congestive heart failure^8–11.

Ethnopharmacology-based research is regarded as a valuable strategy for the discovery of new agents with therapeutic potential^12,13. In recent years, due to their milder action and lower adverse effects, ethnopharmacological remedies are becoming more popular as an alternative treatment for inflammation^14–16. Consequently, there is a strong need for natural products with minimum side effects. Data showed that plants have a wide range of therapeutic properties, including anti-inflammatory properties^17,18.

Peperomia pellucida (L.) Kunth is a member of the Piperaceae family. It is a herbaceous plant with succulent stems, shiny, heart-shaped, new leaves, and tiny leaves that are mostly found in America, Africa, and Asia¹⁹. Peperomia pellucida is known for its variety of pharmacological properties. Traditionally, Peperomia pellucida has been utilized in the folk medicine of several pantropical countries to treat a wide spectrum of ailments and diseases such as abscesses, boils, skin wounds, and eye inflammation (conjunctivitis)²⁰, gastrointestinal disorders, respiratory tract disorders, and skin diseases²¹.

Screenings for phytochemicals on the plant of Peperomia pellucida have revealed the presence of various pharmacologically active principles of medical importance including flavonoids, alkaloids, saponins, tanin, phenolic compounds, phytosterols and steroids. Several essential oils are also found in Peperomia pellucida., mainly dillapiole^22–24. Recent in vitro data have provided new insights into the numerous variables that different subclasses of flavonoids can modulate at various stages of inflammation²⁵. The flavanol quercetin was discovered to suppress COX 2 mRNA expression in rat paw pouch exudate cells, suggesting that quercetin's anti-inflammatory action may be due in part to suppressing COX-2 up-regulation²⁶.

However, Cox-2 inhibitory effect of Peperomia pellucida ethanol extract in vivo have not been described previously. Thus, in the current study, we explored the effect of ethanol extract Peperomia pellucida on anti-inflammation activity in carrageenan-induced rat paw edema and the COX-2 inhibitory effect in vivo.

MATERIALS AND METHODS:

Materials:

Rat PTGS2/COX-2 (Prostaglandin G/H synthase 2) ELISA Kit was purchased from Fine Biotech Co., Ltd. (Wuhan, China). Celecoxib was purchased from Pfizer Inc. (New York, NY, US). Carrageenan was obtained from Sigma-Aldrich (St. Louis, MO, USA) and other reagents were purchased from Bratachem (Indonesia).

The Peperomia pellucida Herb were collected from Pauh Tengah, Kenagarian Sicincin, 2X11 Enam Lingkung, Padang Pariaman, West Sumatera, Indonesia. The Peperomia pellucida Herb were identified by Dr. Nurainas, a botanist at Herbarium of Andalas University, West Sumatera, Indonesia.

Preparation of The Ethanol Extract of Peperomia pellucida (EEPp):

The Peperomia pellucida was sun-dried. The dried Peperomia pellucida (290g) was powdered using a conventional grinder. The powdered materials were then soaked in Ethanol (70%) for 24 hours by stirring at room temperature. The materials were filtered after 24 hours. The procedure was repeated three times. The filtrates were mixed and concentrated under vacuum using a rotary until a brownish semisolid extract (67, 54g) was obtained, free of solvent. The extract was kept cold for further pharmacological testing.

Experimental Animal:

18 adult male Wistar rats with body weights of 180–220 g and aged 2-3 months were obtained from West Sumatera animal houses were used for this study. Animals were housed and cared for in standard conditions with 12h light/dark circle and were fed with a standard pellet diet and water ad libitum. All the animals were acclimatized for a minimum period of 1 week prior to the experiment. After 1 week, animals were randomly selected for different experimental groups (3 animal/ group) and used for the in vivo determination of anti-inflammatory activity. The rats were deprived of food, but not water, for 18–20hours before an experiment. The protocol of this experiment was approved by The Committee of The Research Ethics of The Faculty of Medicines, Andalas University (permit No. 326/KEP/FK/2020).

Evaluation of Anti-Inflammatory activity:

The anti-inflammatory activity was further examined by the carrageenan-induced rat paw edema method according to the method of²⁷. The experimental groups consisted of 18 rats split into six groups, Group I: Negative control (Na. CMC 0,5% p.o only), Group II: positive control (Carrageenan 1% s.c ), Group III: Carrageenan 1% s.c + EEPp (100mg/kg BW p.o), Group IV: Carrageenan 1% s.c + EEPp (200mg/kg BW p.o), Group V: Carrageenan 1% s.c + EEPp (400mg/kg BW p.o), Group VI: comparative group (Carrageenan 1% s.c + Celecoxib 9mg/kg p.o) were given 1h before the injection of carrageenan. By injecting 0.1ml of 1% carrageenan in 0.9% saline onto the right hind paw of the rat, edema was caused. After 1h, 0.1ml 1% carrageenan was injected subcutaneously into the subplantar area of the right hind paw of each rat except those in Group I. Edema volume was determined every 1hour for up to 6 hours after carrageenan administration. The paw volumes were measured by a plethysmometer. The volume difference between before and after the right paw injection was measured to obtain the results. The inflammation degree of the paw and the rate of edema inhibition were calculated as follows:

% Edema inhibition = (Vc −Vt) × 100/ Vc;

Vc and Vt are the average edema volumes of the control and test groups, respectively. The animal blood was collected in a heparin-coated tube at the end of the third hour. Groups I–VI was used to determine the activities of COX-2.

Evaluation of COX – 2 Inhibitory Activity:

Rat serum was prepared at the 3rd hour after induction of Carrageenan 1%. EEPp (100mg/kg BW, 200mg/kg BW, and 400mg/kg BW) was used for inhibition studies.The ability of the test compound to inhibit COX-2 was determined by using the Enzyme-Linked Immunosorbent Assay (ELISA) kit according to the manufacturer’s instructions. The product of this enzymatic reaction produced a distinct yellow color, determined by spectrophotometrically (Microplate reader) at 450nm.

Statistical Analysis:

The statistical software SPSS version 25 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. Data were analyzed using one-way ANOVA followed by Duncan’s multiple range test. p<0.05 was considered significant.

RESULT:

Anti-Inflammatory activity:

In the present study, the edema was measured for 6h after carrageenan injection. EEPp given at dose 100 mg/kg BW, 200mg/kg BW, and 400mg/kg BW were effective in inhibiting the induced paw edema (Table 1).

Table 1 shows the effect of EEPp and standard drug as compared to carrageenan control at different hours in the carrageenan-induced paw edema model. From Table 1, a significant anti-inflammatory activity of EEPp could be confirmed through paw edema inhibition by 34, 93%, 46,61% and 64, 04% after 6 h, at 100, 200, and 400 mg/kg dosage, respectively, while celecoxib diminished paw edema by 66, 67% at the same time. These results demonstrate that the EEPp (100, 200, and 400mg/kg) significantly inhibited the inflammatory processes induced by the injection of carrageenan (p<0,005). These findings indicate that the EEPp posses potent anti-inflammatory properties.

COX-2 Inhibitory Effect:

The COX-2 Inhibitory effect of EEPp could be confirmed through COX-2 inhibition by 10,87%, 47,04 % and 56% after 3 h, at 100, 200, and 400mg/kg dosage, respectively, while celecoxib diminished paw edema by 57, 02% at the same time. These findings show that the EEPp (200, and 400mg/kg B.W) significantly inhibited COX-2 (p<0,005). it was obvious that the EEPp (200, and 400mg/kg BW) ware the most potent COX 2 Inhibitory effect dose (Table 2)

Table 2: COX-2 Inhibitory Effect of EEPp

	Groups	Dose (mg/kg B.W)	Percentage (%) of Cox-2 inhibition^a
I	Negative control (Na. CMC 0,5%)	-	-
II	Positive control (0,1 mL Carrageenan 1% )	-	-
III	EEPp	100	10,87
IV	EEPp	200	47,04*
V	EEPp	400	56*
VI	Celecoxib^b	9	57,02*

^aData are expressed as the mean of Three observations (n = 3), ^bUsed as comparative group

* Significant difference compared to the positive control (P < 0.05)

DISCUSSION:

Carrageenan-induced paw edema is a valuable phlogistic tool for studying systemic anti-inflammatory drugs. This test is sensitive to the majority of anti-inflammatory drugs that are clinically effective and is divided into two phases. The first phase, which occurs within 1-2hours of injection of carrageenan, is caused by serotonin release and an increase in bradykinin, histamine, and prostaglandin in the inflammatory location. The second phase occurs 3-5hours after injection of carrageenan and is associated with kinin and prostaglandin production and release in the inflamed area^28,29. Throughout the second phase, The macrophages are known to produce greater portions of interleukin-1 (IL-1) which triggered a rise in the accumulation of polymorphic nuclear cells (PMNs) at the area of inflammation. The activated PMNs then start releasing lysosomal enzymes and active oxygen species, causing connective tissue destruction and paw swelling³⁰.

In the present study, the EEPp (100, 200, and 400 mg/kg) significantly inhibited the inflammatory processes. This is indicated by the ability of EEPp to significantly decrease rat paw swelling as shown in Tables 1. These results further corroborate previous studies that the aqueous extract of Peperomia pellucida showed antiedematogenic effect³¹. Petroleum ether, chloroform, and methanol extract showed their ability to reduce rat paw edema³². Other studies reported that the extract of Peperomia pellucida showed significant analgesic activity³³. and the leaves of P. pellucida at a dose of 80mg/kg body weight significantly reduced the elevated body temperature of the rabbit³⁴. Pain receptors are found throughout the body and are sensitive to mechanical, thermal, toxic chemical, or inflammatory mediators³⁵. The genesis of pain is inflammatory in most cases, regardless of the aetiology, thus targeting inflammation and pain together is an important concept in pain management³⁶. Because inflammation and pain are linked, several drugs have both analgesic and anti-inflammatory properties.

COX and 5-LOX are two key enzymes that catalyze the emergence of inflammatory mediators. Cox inhibitors are the main strays of current therapy aimed at modulating pain, inflammation, and fever control³⁷. Many COX-2 or 5-LOX inhibitors have been developed as anti-inflammatory drugs; however, some have been withdrawn from the market, suggesting the need for inhibitors with minimal side effects³⁸. Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their analgesic effects by inhibiting cyclooxygenase activity, antipyretic, anti-inflammatory, and antithrombotic effects^39,40. In the present study, EEPp significantly inhibited COX activity in rats at the third hour in vivo when compared to the control. These findings indicate that the EEPp posses potent anti-inflammatory properties. These results further corroborate previous studies that Peperomia pellucida showed anti-arachidonate 5-lipoxygenase (Anti-LOX) activity⁴¹ and anticancer⁴². The anti-inflammatory potential of EEPp may be due to the presence of active phytoconstituents such as flavonoids. The previous report describes flavonoids can reduce the risk of atherosclerosis and atherothrombotic disease and several other inflammatory diseases^43–45. The flavanol quercetin was found to suppress the expression of COX 2 mRNA in rat paw pouch exudates cells, indicating that quercetin's anti-inflammatory action may be due in part to suppressing COX-2 up-regulation⁴⁶. Flavonoids can interfere with the oxidative synthesis of (Arachidonic Acid) AA from phospholipids and decrease the downstream production of inflammatory metabolites from AA metabolism, oxidative damage, and initiation of inducible pathways of Inflammation due to their powerful antioxidant capacity^47,48. Other studies have demonstrated that flavonoids with antioxidant properties can reduce the cellular conversion of AA to MDA (Malondialdehyde) in patients with chronic inflammation⁴⁹. Radical scavenging activities of phenolic and polyphenolic compounds have been shown in previous studies^50–52. There are also many studies on anti-inflammatory activities of plant extracts that contain flavonoids^53,54. In addition, Peperomia pellucida possessed vast potential as a medicinal drug, especially in cancer treatment and antioxidant^55,56. There is a chance that the significant antioxidant capacity and flavonoid content of the extract being used have contributed to the anti-inflammatory effect.

This study is the first report on the potent COX-2 inhibitory properties of the ethanol extract of Peperomia pellucida. Obtained results indicate that the inhibition of prostaglandin synthesis mediated via the arachidonic acid pathway could be responsible for the biological effects of this natural compound.

CONCLUSION:

From the results of the present investigation, it can be concluded that the EEPp possesses significant and promising anti-inflammatory activity. The mechanism of anti-inflammatory action is thought to be mediated by COX-2 inhibition. With the attained results, it could be speculated that EEPp might be a potential alternative anti-inflammatory agent and the results presented also suggest the need for further research on the COX-1 inhibitory effect.

CONFLICT OF INTEREST:

The authors declare that there are no conflicts of interest.

ACKNOWLEDGMENTS:

The authors are grateful to all colleagues at The Department of Pharmacology and Clinical Pharmacy, School of Pharmaceutical Science Padang, for helpful discussions.

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Received on 19.08.2022 Modified on 17.10.2022

Research J. Pharm. and Tech 2023; 16(8):3727-3732.

DOI: 10.52711/0974-360X.2023.00615

Groups		Dose (mg/kg BW)	Percentage (%) of paw edema^a						Percentage (%) of paw edema inhibition
Groups		Dose (mg/kg BW)	1st hour	2nd hour	3rd hour	4th hour	5th hour	6th hour	Percentage (%) of paw edema inhibition
I	Negative control (Na. CMC 0,5%)	-	0 ± 0.00	0 ± 0.00	0 ± 0.00	0 ± 0.00	0 ± 0.00	0 ± 0.00	-
II	Positive control (Carrageenan 1%)	-	86 ±0.09	52 ±0.04	74 ±0.04	66 ±0.04	66 ±0.04	66 ±0.04	-
III	EEPp	100	86 ±0.11	60 ±0.00	52 ±0.05	34 ±0.05	20 ±0.00	20 ±0.00	34,93*
IV	EEPp	200	72 ±0.04	46 ±0.04	40 ±0.00	20 ±0.00	20 ±0.00	12 ±0.04	46,61*
V	EEPp	400	52 ±0.04	20 ±0.00	34 ±0.04	20 ±0.00	20 ±0.00	6 ±0.04	64,04*
VI	Celecoxib^b	9	50 ±0.14	32 ±0.00	26 ±0.04	13 ±0.00	13 ±0.00	5,6 ±0.04	66,67*