1. INTRODUCTION:

Rubiadin, is a anthraquinone that has been isolated from Rubia cordifolia Linn roots belonging to family- Rubiaceae and good number of activites have been reported regarding its isolation, characterization, analysis and formulation.^1-7 The plant Rubia cordifolia bears anti-inflammatory, immunomodulatory, anticonvulsant and anti-tumor activities.^8-10 Rubiadin has been evaluated by our research groups for its analgesic activities.¹¹In view of the above points, Rubiadin was evaluated for its role in modulation of chronic pain.

2. MATERIAL AND METHODS:

2.1 Drugs and reagents- The Rubiadin [chemically it is 1,3-dihydroxy-2-methylanthracene-9,10-dione] purchased from (Product code: R004, Lot. no.: T19D079; CAS No: 117-02-2) Natural Remedies Pvt. Ltd., Bangalore. Rubiadin purity was determined by the manufacturer via HPLC certifying purity above 94.80%. CFA purchased from Sigma (St Louis, MO). Indomethacin purchased from Elder pharma. Analytical grade chemicals and reagents were used in this study.

2.2 Animals- Swiss female mice (weighing in between 25 to 35 g were utilized with freely available food and water. The mice kept under standard environment and fed with standard pellet diet. Food and water were given freely throughout the entire experiments. Approval was obtained from the Institutional Animal Ethics reference number- RCP/18-19/CPCPSEA/P-20. The experiments were carried as per CPCSEA ethical guidelines.

2.3 Complete Freund’s adjuvant injection- Anaesthetized mice given CFA; 1mg/mL subcutaneously in right paw plantar surface (ipsilateral paw). The inflammation was evaluated by measuring paw volume for edema and mechanical hypersensitivity 24 hours prior CFA injection.

2.4 Behavioral evaluation of chronic pain signs- Nociception was assessed by applying mechanical and cold stimuli, to mice hind paw by placing individually in meshed elevated platform to allowing access to paws ventral surface.^12-13 Animals habited for environment and chamber testing for at least 10–15 minutes, or until exploratory behavior cessation, before initial assessments. Basal responses to mechanical or cold stimuli were evaluated prior CFA injection and after treatments between 9.00 and 6.00 PM.

2.5 Mechanical hypersensitivity evaluation- Von Frey filaments calibrated to 0.1, 0.25, 0.50, 1, 2, 3 and 4g, forces applied to the middle region of the plantar surface of hind paw for 5 seconds, or until the animal displayed a nocifensive response concluded as paw licks, lifts and/or shakes. Initially testing was started with 0.1g filament.¹³ In absences of clear paw withdrawal, incrementally stronger filaments were then presented consecutive till mice found eliciting such responses.

2.6 Cold hypersensitivity evaluation- Briefly with help of syringe, 20µL acetone is applied on mid plantar surface of paw. The time spent licking or the paw shaking, from acetone evoked cooling is noted for 5 minutes after applying acetone, utilizing stopwatch which is used for indication of nocifensive index response for cold stimuli. Significance of these indexes interprets hypersensitivity to cold.

2.7 Paw edema evaluation- Previously reported method is used to analyze Rubiadin effects on paw caused due to intraplantar CFA injection. The degree of paw edema inflammation is measured utilizing VJ Instruments digital plethysmometer at half hourly time-points and expressed as milliliters as the difference between the paws volumes such as between CFA injected inflamed paw called as ipsilateral paw and the other noninjected as contralateral paw.

2.8 Induction of inflammation, pain and Rubiadin treatment protocol- The details treatment protocol in five groups of mice is follows:^12-13

i. Control administered with 0.2mL DMSO intraperitoneally;

ii. CFA inflamed receiving intraperitoneal 0.2mL of DMSO.

iii. CFA inflamed receiving intraperitoneal, standard Indomethacin10mg.

iv. CFA inflamed receiving intraperitoneal 100 mg/kg Rubiadin,

v. CFA induced inflamed mice receiving intraperitoneal 200mg/kg Rubiadin.

Inflammatory pain induced by CFA was induced in all groups except for the control vehicle group as described in above section by a single CFA injection in right paw. Acute responses evaluated at 24 hours prior CFA injection, mice given Indomethacin, Rubiadin (100 and 200mg, i.p.) or vehicle. Mechanical hypersensitivity, cold sensitivity of Rubiadin was evaluated at subsequent time-points along with paw edema measuring. For investigating Rubiadin effects on chronic mechanical hypersensitivity, Rubiadin administered twice a day mice (12 hours apart) for 7 days period. This treatment being interrupted on day five and restarted after 3^rd day for possible investigation of tolerance development. The nociceptive evaluation done 3 hours post daily first treatment such as time when maximal inhibitory response was seen after acute Rubiadin treatment.

2.9 Statistical analysis- The statistical analysis between groups determined by ANOVA (analysis of variance) following Student test. P values less than 0.05 considered significant (P<0.05). The results presents mean±SEM (standard error of mean).

3. RESULTS:

3.1 Rubiadin effect on Complete Freunds adjuvant-induced inflammatory hyperalgesia:

This study investigates Rubiadin antiallodynic effects in persistent CFA inflammatory pain model of nociception in mice. CFA produced inflammation lasting from hours, days to weeks and mimicking sub-acute pathogenic conditions of rheumatoid arthritis along with chronic pain signs.

3.2 Rubiadin effect on mechanical allodynia:

Mice received Rubiadin (100 and 200mg intraperitoneally) 30 minutes prior testing, significantly reducing by CFA inducted mechanical allodynia which was initiated 60 minutes post administration of Rubiadin and maintained untill 12 hours. The marked effect being observed around 8 hours post Rubiadin (200mg/kg) administration, when inhibition reached 91.14%.

3.3 Rubiadin effects on cold hypersensitivity:

The results indicate Rubiadin can reduce CFA induced cold hypersensitivity. The best anti-allodynic effects in mice were observed in time interval of 4 to 10 hour period post Rubiadin administration. The maximal inhibition of 85.54% at 5 hours for Rubiadin 100mg/kg and 93.05% at 6 hours for Rubiadin 200mg/kg was observed.

3.4 Rubiadin effects on paw volume on acute treatment- CFA injection initiates increase in paw volume 24 hours post CFA injection. Rubiadin significantly reduces paw edema at two studied doses 100 and 200mg post 1-2 hours, with highest percent of reduction between 7–8-hour period for details refer figure 1.

Fig. 1 Effects Rubiadin on paw edema in CFA injected mice. [Acute effects of Rubiadin after intraperitoneal administration on mechanical hypersensitivity in mice 24 hours prior CFA injection. Each point indicate mean ± SEM in paw volume in milliliters of the paw of ipsilateral paw before and after injection of CFA at indicated time points. P < 0 .01 and P < 0.05 are levels of significance of Rubiadin compared to only CFA treated mice.

3.5 Rubiadin effects on chronic mechanical allodynia- Prolongatory intraperitoneal twice a day Rubiadin treatment reduce significantly mechanical hypersensitivity induced by CFA (peaked on 5^th day with maximum inhibition of 78.22 % for Rubiadin 200 mg and 57.47% for 100mg/kg please refer figure 2 for in detail analysis. Treatment of Rubiadin was interrupted for 3 days reestablishing mechanical hypersensitivity. Treatment of Rubiadin was restarted from 9th day. It was noted that Rubiadin once again reduced mechanical hypersensitivity significantly, which excludes the possible tolerance development of after Rubiadin treatment.

Fig. 2 Chronic Effects Rubiadin on mechanical hypersensitivity in CFA injected mice prior 24 hours. [Acute effects of Rubiadin after intraperitoneal administration on mechanical hypersensitivity in mice 24 hours prior CFA injection. Each point indicate days with mean ± SEM in grams of the paw withdrawal threshold in response to stimulation of the ipsilateral paw before injection of CFA (baseline) or prior indicated times. On the 6^th, 7^th and 8^th day Rubiadin dosing is stopped for evaluating resistance. P < 0 .01 and P < 0.05 are levels of significance of Rubiadin compared to only CFA treated mice.

4. DISCUSSION:

4.1 CFA induces chronic hyperalgesia:

CFA plantar injection induced inflammatory pain in paw. An effect mediated by peripheral as well as central mechanisms at spinal cord level and involves inflammatory mediators like excitatory amino acid like glutamate and neuropeptides from C fibres, including bradykinin receptor (B1) sensitization contributing to hyperalgesic response. CFA induced inflammatory hyperalgesia lead to releasing nociceptive inflammatory mediators increasing sensitivity to peripheral as well as central sensory pathways.^11-16 Also increased glutamate levels and also rise in cytokinesis including interleukin (IL-1β) and tumour necrosis factor -α in spinal cord dorsal horn neurons, contributing towards their hypersensitivity. All this occurs by direct opening of ion channels, activating intracellular mediators and nitric oxide induction. Bradykinin and prostaglandin E2 receptors are also activated. All these events contribute to transmit and maintain hypersensitivity of chronic pain.^11-16

4.2 Anti-hyperalgesia of Rubiadin:

The present data reveals Rubiadin dose dependent potential for attenuation of CFA-induced inflammation and pain. The higher dose of Rubiadin alleviates prominently early phase edema and pain (24 hours) along with better recovery in later stages of chronic hyperalgesia. Nothing significant toxic or side effects were noted in the current studied two doses. It was observed that Rubiadin antihyperalgesic effects in CFA induced model can be correlated to its effects post inflammatory hyperalgesia such as 24 hours and 5 days after injection of CFA. This study shows Rubiadin given systemically in mice is efficacious to inhibit mechanical aa well as cold hypersensitivity, also it reduced paw edema initiated by CFA. Anti-nociceptive effect of Rubiadin, on mechanical sensitivity evidenced in early phase at half hour which lasts until end of experimentation such as ten hours. Its recognizable that persistency of sensitivity of CFA is mediates inflammatory mediatory components like cytokines, glutamate, nitric oxide, prostanoids, histamine, serotonin and other subsequent immunological complimentary agents. Mainly anti-nociceptive potential of Rubiadin may be associated with a anti-inflammatory action locally, as Rubiadin reduces paw edema induced by CFA.

4.3 Chronic pain modulatory effects of Rubiadin: Acute pains substantially differ from Chronic one in terms of its persistency, adaptively neuroplasticity.^13,15

This study confirms first time Rubiadins ability to produce significant antinociception in CFA induced chronic inflammation and pain in mice. Studies from our laboratory show Rubiadin might initiate its antinociceptive responses by involving dual peripheral plus central mechanistic antinociception.^1,16 It’s crucial to highlight Rubiadin anti-allodynic potential is non susceptible towards tolerance, as Rubiadin can maintain its action efficacy after repeated administered intraperitoneally. The present data favors a probable therapeutic efficacy of Rubiadin to treat chronic pain. Even though clear mechanism underlying the anti-nociceptive effects of Rubiadin remains unclear and it might be assumed that the inhibitory indulgence in prostanoid pathway could be accountable for its antinociceptive effects noted in present model but needs future in detail investigations are a must.

For explorations of varieties of mechanisms, computer aided-drug designing techniques can be further utilized in relation with our study.^17-23

4. CONCLUSION:

This study supports the probable anti-hyperalgesic action of Rubiadin that could be mediated due to its analgesic as well anti-inflammatory capacities. However, further in-depth studies are encouraged for its conclusive effectiveness therapies for chronic pain.

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Received on 18.11.2021 Modified on 04.02.2022

Research J. Pharm. and Tech 2023; 16(1):31-34.

DOI: 10.52711/0974-360X.2023.00006