Computational Evaluation and Anti-inflammatory and Analgesic Activities of Nebrodenside A Isolated From Dodonaea viscosa

Abstract

Nebrodenside A was isolated from the plant Dodonaea viscosa and its chemical structure was elucidated by spectroscopic methods. Molecular docking simulation predicted its strong ability to be tested as a potential anti-inflammatory compound. Carrageen-induced paw model and formalin test were utilized for the assessment of anti-inflammatory and analgesic potential. Nebrodenside A was found to be significantly active in reducing inflammation, when compared with diclofenac as a standard drug. Similarly, the compound also possesses substantial analgesic activity in the formalin-induced writhing test. Thus, nebrodenside A revealed a robust potential to be developed as a possible leading medicinal agent for better management of pain and inflammation.

Dodonaea viscosa (L.) species belong to the Sapindaceae family (native plant to Australia) and possess various medicinal activities such as analgesic, anti-inflammatory, and antimalarial. ¹ In addition, the extract of D. viscosa can be used as local anesthetic, muscle relaxant, antifungal, anti-ulcerogenic, and antiparasitic agent. ² Recent research has been in progress to isolate such drugs from the natural sources, especially, medicinal plants which have high efficacy and low side effects. In spite of recent advancements in therapeutic management of pain and inflammation, potent drugs with lower side effects are needed as therapies for several agonizing inflammatory diseases. ^3,4 In the past, pharmacologically active analgesics from plants such as aspirin and morphine were discovered ^3,5 ; but these drugs are associated with adverse effects. So, new effective yet safer drugs from natural sources, especially medicinal plants, need greater attention and efforts. In this context, our research group probed nebrodenside A (Figure 1), a phenolic compound, for its palliative and anti-inflammatory activities in murine models, with the idea to discover a novel lead compound for better administration of pain and inflammation. Nebrodenside A was isolated in grams (1.3 g) from D. viscosa and had good spectroscopic matching with the compound previously isolated from Ephedra nebrodensis. ⁶

Figure 1

Chemical structure of nebrodenside A.

Inflammation is a fundamental biological response of the body which is linked with several pathological disorders including pain and fever. Different therapeutic targets are involved in inflammation signaling, ⁷ in which cyclooxygenase-2 (COX-2) is very significant as it regularizes the prostaglandin biosynthetic pathway. In addition, COX-2 inhibitors control signs and symptoms of inflammation such as redness, swelling, pain, and fever, but have adverse side effects, including gastric ulceration. ⁸ In this context, the isolated compound was computationally viewed through molecular docking simulations in comparison to the standard marketed drug diclofenac.

The standard diclofenac compound was docked with the COX-2 receptor protein (PDB ID: 5JVZ) and this resulted in a strong binding, with four hydrogen binding interactions by the carboxylic group of the compound. The carbonyl group of the diclofenac accepts hydrogen bond from asparagine 39 and cystine 37 of the COX-2 receptor protein. The hydroxy group of the carboxylic group of the diclofenac donates a hydrogen bond to proline 155 and accepts a hydrogen bond through its hydroxy oxygen atom from glutamine 462 of the receptor protein, as presented in Figure 2. The calculated binding energy of diclofenac sodium with COX-2 was −17.55 kcal/mol.

Figure 2

The left panel shows the 2D picture of the binding pocket of COX-2 receptor protein interaction with diclofenac compound through hydrogen bonding interaction whereas the right panel is a 3D view of the same binding pocket. COX-2, cyclooxygenase-2.

When nebrodenside A was docked to the COX-2 receptor protein (PDB ID: 5JVZ), it bound in the same fashion inside the same pocket like the standard diclofenac compound. The phenol moiety of nebrodenside A forms hydrogen bonds with cystine 36 and asparagine 39 of COX-2 receptor protein as shown in Figure 3. The hydroxy group is the hydrogen bond donor and the carboxyl groups of cystine 36 and asparagine 39 of COX-2 are the hydrogen bond acceptors. The two hydroxy groups at carbon-3″ and carbon-6″ positions of the glucose residue of nebrodenside A also act as hydrogen bond donors. The C-3″ hydroxy group of the sugar makes a hydrogen bond with glycine 45 whose carboxyl group acts as a hydrogen bond acceptor, whereas the C-6″ hydroxy group donates a hydrogen bond to the carboxyl group of alanine 151 of the COX-2. The total binding energy of nebrodenside A on docking with COX-2 was −23.4 kcal/mol. From the collective binding free energy value, it is obvious that these hydrogen bonding interactions are very strong that nebrodenside A interacts strongly with the COX-2 receptor.

Figure 3

Molecular interactions of the COX-2 receptor protein (PDB ID: 5JVZ) with nebrodenside A. The left panel shows the binding pocket of COX-2 protein interacting with nebrodenside A through hydrogen bonding interaction. The right panel is the 3D picture of the same binding pocket. COX-2, cyclooxygenase-2.

In addition, standard diclofenac compound and tested nebrodenside A were also docked with the COX-1 receptor protein (Supporting data). These docking results showed that nebrodenside A is more selective toward COX-2 protein (PDB ID: 5JVZ) than COX-1 protein (PDB ID: 3KK6). These results agree with the in vivo results, which show that the drug is effective at later time and that it inhibits the production of prostaglandins which are produced by COX-2.

This estimation and prediction led to the in vivo experimental analgesia of nebrodenside A in carageenan-induced paw edema model of inflammation, as well as in formalin-induced pain model. ⁷ Nebrodenside A produced a significant anti-inflammatory action in vivo by controlling the two-stage inflammation made by carrageenan as presented in Table 1.

Table 1

Anti-inflammatory Effects of Tested Compound on Carrageenan-Induced Paw Edema in Rats.

Treatment	Dose (mg/kg)	Increase in paw volume (mm)
		Early phase (3 h)	Late phase (5 h)
Control	-	5.83 ± 1.06	5.9 ± 1.24
Test compound	10	4.79 ± 1.22	4.53 ± 1.39*
Test compound	20	4.07 ± 0.98*	3.72 ± 1.06*
Test compound	40	3.21 ± 0.83*	2.98 ± 0.92*
Diclofenac	5	2.94 ± 0.78**	2.31 ± 0.66**

Values are mean ± SD, n = 6, *P < 0.05 and **P < 0.01 compared to control group.

The initial part (90-180 minutes) of the inflammation is the result of physiological agents like histamine, serotonin, and related biochemical compounds that are released by the specialized cells in the body. The last phase (270-360 minutes) of edema tempted by carrageenan agent is grouped by the highest volume of hind limb, where the edema is at its peak, and by the release of prostaglandins and other inflammatory mediators like kinin substances, for example, proteases and lysosomes. ⁹ These inflammatory intermediaries are responsible agents that cause swelling and other edematous-related reactions. All animals were found devoid of any statistically considerable mortality and morbidity, after 48 hours of administration of the test compound in the mentioned dose. The valuable results of this tested compound are almost similar and it possibly works by the same anti-inflammatory mechanism as diclofenac (Table 1) that covers the inhibition of inflammation process induced by carrageenan. ⁹

The formalin-initiated paw licking model embraces both the initial and late phases. ¹⁰ The early phase (immediately after injection) seems to be caused by C-fiber activation as a result of peripheral stimulus. ¹¹ The late stage (starting approximately 20 minutes after formalin treatment) depends on a number of factors, which include an inflammatory reaction, activation of N-methyl-d-aspartate (NMDA) and non-NMDA receptors, and the nitric oxide pathway, ^11,12 in the peripheral tissue and the physiological changes in the dorsal horn of the spinal cord. ^10,12 In the current analyses, nebrodenside A showed strong activity in the early stage at a dose concentration of 20 and 40 mg/kg (Table 2) showing a full inhibition of C- fiber in the initial stage.

Table 2

Analgesic Effect of Tested Compound on Formalin-Induced Noxious Pain in Mice.

Treatment	Dose (mg/kg)	Score of pain severity
		Early phase (0-10) min	Late phase (15-30) min
Control	-	2.8 ± 0.2	2.8 ± 0.3
Test compound	10	1.6 ± 0.1*	1.5 ± 0.2*
Test compound	20	1.1 ± 0.1*	0.9 ± 0.1*
Test compound	40	0.6 ± 0.1*	0.5 ± 0.3*
Diclofenac	5	0.2 ± 0.1**	0.2 ± 0.2**

Values are mean ± SD, n = 6, *P < 0.05 and **P < 0.01 compared to control group.

Nebrodenside A lowers the response time in a dose-dependent manner in the late stage which might suggest that the compound resulted in some inhibition of NMDA and non-NMDA receptors. ¹⁰ It was concluded that nebrodenside A has a promising analgesic property which is probably marginally facilitated via the blockade of prostaglandin formation along with a central inhibitory mechanism and it might be of prospective advantage for pain management. ¹⁰ Furthermore, these observations on antagonistic action over inflammation showed that such compounds should be comprehensively evaluated in vitro and in vivo before developing them into novel potential contenders for the inhibition of inflammation.

Experimental

Molecular Docking Analysis

The structural coordinates of COX-2 having PDB code 5JVZ was taken from the RCSB Protein Data Bank. ^13,14 The non-protein atoms and water molecules in the PDB file were removed and the addition of H-atoms was done through molecular operating environment. Initially, energy minimization of the enzyme was done utilizing the AMBER99 force field before molecular docking. Nebrodenside A was then constructed and the missing H-atoms were added using the protonate 3D module and its structure was energy minimized using the MMFF94 force field presented in Parlar et al ¹⁵ and Soyer et al. ¹⁶

Animal Models for in Vivo Studies

For in vivo studies, adult Wistar rats having approximate weight in the range of 180 to 260 g and Swiss albino mice with weight range of 18 to 25 g were used. These murine models were kept at the Animal House Laboratory of the National Institute of Health, Islamabad, Pakistan. ¹⁷ Murine species were kept at normal room temperature of 25°C and light/dark cycles (12/12 hours) throughout their life. All the tests were performed as per the guidelines mentioned in the guide for the care and use of laboratory animals issued by the US Department of Health and Human Services, Institute of Laboratory Animal Resources, Washington, DC. ¹⁸

Antinociceptive Activity

The method of Dubuisson, ¹⁹ with amendments by Tjølsen et al, ²⁰ was utilized for the antinociceptive activity. The murine model was taken and the test samples were administered 30 minutes before the formalin test, followed by 5 µL of formalin (2.5% formaldehyde) injection into the plantar surface of the rat hind paw; behavioral responses detected were recorded as scores in the following manner: (1) rat walking or standing on injected paw, paw partially elevated, total elevation of injected paw; (2) injected paw licking or biting; (3) scores of the first 10 minutes after formalin will be recorded as the first phase of analgesia and the period between 15 and 60 minutes as the late phase of pain.

Anti-inflammatory Activity

For determining the possible anti-inflammatory action, carrageenan was used to induce paw edema as per the available reported protocol with some modification. ⁹ Serious inflammation was made by subplantar injection of 1% suspension of carrageenan solution (100 µL) with 2% gum acacia solution as a suspension mediator. Different treatment groups were compared to find out the anti-inflammatory result of test and control samples. These inflammatory intermediaries are responsible agents that cause swelling and other edematous-related reactions. All animals were found devoid of any statistically considerable mortality and morbidity. ²¹

Statistical Analysis

All the results in these observations were expressed as a mean ± SD. The Student’s t-test was used to analyze the results obtained by comparison between groups and variance in them was tracked by the famous Dunnett’s test for multiple comparative studies and P < 0.05 was taken substantial in all data calculations. ⁷

Plant Collection and Isolation

The plant material was collected from the hills of Kurram Agency, Khyber-Pakhtoonkhwa, Pakistan. The identification of the plant and extraction, fractionation, and isolation of the secondary metabolites were reported. ¹

Compound Name

Nebrodenside A

White crystalline powder (1.3 g)

[α]²⁵ _D: +60 (c 0.3, CD₃OD)

UV (MeOD) λ _max (log ε) nm: 225, 287

IR max (KBr)/cm: 3400, 2973, 2928, 2907, 1644, 1606

¹H NMR (C₂D₆CO, 300 MHz): δ 6.82 (1H, d, J = 2.4 Hz, H-2), 6.76 (1H, t, J = 8.7, 2.4 Hz, H-6), 6.71 (1H, d, J = 8.4 Hz, H-5), 3.41 (2H, m, H-1′), 5.34 (1H, t, J = 6.0 Hz, H-2′), 1.71(3H, s, H-4′), 1.69 (3H, s, H-5′), 4.76 (1H, d, J = 7.5 Hz, H-1″), 3.34 (m, 1H- 2″), 3.38 (1H, m, 3″), 3.28 (1H, m, H-4″), 3.40 (1H, m, H-5″), 3.70 (1Ha, dd, J = 11.7, 5.8, H-6″), 3.87 (1Hb, dd, J = 11.4, 2.1, H-6″).

¹³C NMR (C₂D₆CO, 75 MHz): δ 152.2 (C-1), 119.2 (C-2), 129.4 (C-3), 150.8 (C-4), 115.9 (C-5), 115.7 (C-6), 29.0 (C-1′), 123.5 (C-2′), 132.6 (C-3′), 17.8 (C-4′), 25.9 (C-5′), 103.0 (C-1″), 74.8 (C-2″), 78.0 (C-3″), 71.5 (C-4″), 77.6 (C-5″), 62.7 (C-6″).

EI-MS m/z (rel. int. %): 178 (100), 123 (51), 73 (19).

ESI-MS (M + Na) m/z: 363.1374 (calcd. for C₁₇H₂₄O₇ 363.1374) FAB-MS (+ve): 340.0.