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ORIGINAL ARTICLE
Year : 2017  |  Volume : 7  |  Issue : 4  |  Page : 88-93

Protective Effects of a Polyherbal Formulation, Freemodex, Against Acute Models of Pain, Inflammation, Arthritis, and Immunosuppression


1 Department of Pharmacology & Toxicology, Zydus Research Centre, Ahmedabad, Gujarat; Department of Pharmacology, Shree Devi College of Pharmacy, Mangalore, Karnataka, India
2 Department of Pharmacology, Shree Devi College of Pharmacy, Mangalore, Karnataka, India

Date of Web Publication6-Nov-2017

Correspondence Address:
Laxit Bhatt
Department of Pharmacology & Toxicology, Zydus Research Centre, Sarkhej-Bavla N.H. No. 8A, Moraiya, Ahmedabad – 382 210, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijnpnd.ijnpnd_31_17

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   Abstract 

Aim: This study was designed to evaluate the analgesic, anti-inflammatory, antiarthritic and immunomodulatory activities of Freemodex. Materials and Methods: Acute models of pain – formalin-induced paw licking, inflammation – egg albumin induced paw edema, arthritis – turpentine-induced arthritis, and immunosuppression – carbon clearance test and cyclophosphamide-induced neutropenia were evaluated using rats and mice. The degree of protection was determined for antiarthritic, analgesic, and anti-inflammatory activities by measuring the paw volume, percentage inhibition, the number of writhing movements, the duration of paw licking in the injected paw, paw linear circumference, and the joint diameter. The estimation of phagocytic index, the total leukocyte count, and the percentage reduction in the neutrophil count were indexed for the evaluation of immunosuppression. Freemodex was administered at 40 and 80 mg/kg to rats and 80 and 160 mg/kg to mice. Results: Dose-dependent reduction in paw licking was observed in the formalin model. Reduction in paw volume was seen in the animals treated with egg albumin and Freemodex. Arthritis was also found to be decreased on the basis of the synovial joint size and phagocytic index. Leukocytes and neutrophils were also normalized in mice. Conclusion: The study’s findings suggest a protective role for Freemodex.

Keywords: Antiarthritic, Freemodex, immunomodulatory, inflammation, pain


How to cite this article:
Bhatt L, Maithani S. Protective Effects of a Polyherbal Formulation, Freemodex, Against Acute Models of Pain, Inflammation, Arthritis, and Immunosuppression. Int J Nutr Pharmacol Neurol Dis 2017;7:88-93

How to cite this URL:
Bhatt L, Maithani S. Protective Effects of a Polyherbal Formulation, Freemodex, Against Acute Models of Pain, Inflammation, Arthritis, and Immunosuppression. Int J Nutr Pharmacol Neurol Dis [serial online] 2017 [cited 2017 Nov 18];7:88-93. Available from: http://www.ijnpnd.com/text.asp?2017/7/4/88/217750


   Background Top


Injury, irritation or infection can cause inflammation and pain. Redness (rubor), heat (calor) and swelling (tumor) are the classical accompaniments of pain. Each of them contributes to the damage of the tissues. Inflammation and inflammatory pain is observed in some diseases like arthritis, where it results into a chronic state. The progression of inflammation and pain associated with it is mediated by different agents known as inflammatory mediators that result into a cascade of inflammatory processes. Agents that prevent the actions of these mediators are widely used in treatment of inflammation, pain and ailments associated with it, such as arthritis. Anti-arthritic treatment consists of: Disease-modifying anti-rheumatic drugs (DMARDs), Biologic response modifiers, glucocorticoids, nonsteroidal anti-inflammatory medications (NSAIDs), and analgesics. However, most of these drugs produce serious side effects including but not limited to: ulceration, gastrointestinal upset, diarrhoea, nodulosis, headache, nausea, hair loss, and thrombocytopenia.[1],[2],[3],[4]

Herbal medicines have been in existence since the prehistoric period. They have been an integral part of therapy in African, Egyptian, Chinese and Indian cultures and most are still used in their original form. Ayurveda is India’s own system of medicine developed solely on natural sources, and herbs have continued to be one of its most powerful healing ingredients.[5],[6],[7],[8] Drug formulations in Ayurveda, as described in the ‘Sarangdhar Samhita’, are either made up of a single drug (animal, plant or mineral source) or of two or more drugs (animal, plant or mineral source or a combination of them) also known as a Polyherbal formulation (PHF). The PHFs are used to gain extra therapeutic effects which results likely due to synergism between effects of several herbs that make up a PHF. PHFs have been widely used in treatment of various ailments in the Ayurveda system of medicine.[9]

Freemodex is a polyherbal formulation developed by Jaffman Pharmaceuticals consisting of a mixture of 34 different herbs: Dwipantaravacha, Musta, Mahayogaraj Guggul, Guggul Purified, Sallaki, Shunti, Lasuna, Ashwagandha, Rasna, Ballataka Purified, Kuchala Purified, Haridra, Amrita, Shatavari, Shilajit, Amalaki, Haritaki, Vibhitaki, Katukaronini, Sameerapannagaras, Mahavidwansras, Ela, Twak, Patra, Maricha, Nirgundi, Neelanirgundi, Gandhaprasarini, Amrit, Bala, Shigru Indravalli, Erand, Lajjalu and Arjuna. These ingredients have different protective pharmacological activities.[10],[11],[12],[13] Freemodex is particularly used in incidences of pain and inflammation. It is also used in treatment of rheumatoid arthritis, an auto-immune disorder that affects the joints.

However, the data on the therapeutic potential of this polyherbal formulation is unavailable. Hence, this study is designed to evaluate the analgesic, anti-inflammatory, anti-arthritic and immunomodulatory activities of Freemodex using various animal models.


   Materials and Methods Top


Animals

Swiss albino mice of either sex weighing 18–25 g and wistar rats of either sex weighing 175–250 g were obtained from the animal facility of Shree Devi College of Pharmacy, Mangalore, India. Animals were maintained in an animal house at 24 ± 5 °C under 12 h light-dark cycles. They were housed in IVC racks with provision for feed and water (ad libitum). Animals were acclimatized for one week to laboratory conditions before starting the experiment. All experiment protocols were conducted according to the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), the Ministry of Social Justices and Empowerment, Government of India. Prior to the commencement of the experiment, approval was obtained from the Institutional Animal Ethics Committee (SDCP/IAEC-05/2012-13).

Drugs under study

Freemodex capsules manufactured by Jaffman Pharmaceuticals were purchased from local pharmacy stores from Mangalore area. The polyherbal formulation from the capsules was emptied and suspended into distilled water. The formulation was administered orally using an oral gavage at doses of 40 and 80 mg/kg to rats and 80 and 160 mg/kg to mice. Commercially available Diclofenac (10 mg/kg, Pfizer) was used as the standard control for anti-arthritic,[14] anti-inflammatory[15] and analgesic[16] models. Distilled water served as vehicle control. Chemicals and reagents were purchased from standard organisations and were of highest analytical grade.

Experimental groups

Animals were randomised into four groups with six animals in each as described in [Table 1].
Table 1: Experimental groups

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Model for pain – formalin induced paw licking test in mice

Formalin (20 μl of 2.5% solution) was injected into the sub-plantar region of the right hind paw and same volume of sterile phosphate buffered saline into the left paw, 1 hour after the administration of study drugs as described in [Table 2]. Index of nociception was determined by measuring the duration of paw licking in two periods: Early Phase, 0–5 mins and Late Phase, 15–30 mins.[17]
Table 2: Effect of Freemodex in egg albumin induced hind paw edema in rats

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Model for inflammation – egg albumin induced hind paw edema in rats

Egg albumin (0.1 ml, 1% in normal saline) was injected into the sub plantar region of the right hind paw. Study drugs were administered to 24 hours fasted mice 1 hour before the induction of inflammation. The linear circumference of the injected paw was measured before and 0.5, 1, 2, 3, 4 and 5 h after the administration of egg albumin. Edema (inflammation) was assessed as the difference in paw circumference between the control and 0.5, 1, 2, 3, 4 and 5h after the administration of egg albumin. The average (mean) edema was assessed by measuring with Vernier Calipers (Sylvac Metrology, Pune, India).[18]

Model for arthritis – turpentine induced arthritis in rats

Baseline recording of the left hind paw volume was made by using a Mercury Plethysmometer (INCO, Ambala, India) on the day of experimentation. Animals were fasted overnight before administration of study drugs [Table 1]. Turpentine oil (0.02 ml) was injected into the synovial cavity of the knee joint of the animals, 30 mins after the administration of study drugs. Diameter of the joint was measured every hour till 6 hours after turpentine oil injection using a calibrated Micrometer Screw Gauge.[19]

Model for immunomodulation – carbon clearance test in mice

Swiss albino mice were administered with study drugs [Table 1] for 10 days. Forty-eight hours after the last dose of the drug, animals were treated with Indian ink (Colloidal carbon, 0.3 ml/30 grams, i.v.) via the tail vein. Blood samples were withdrawn from each animal through retro-orbital plexus at an interval of 0 and 15 min after the ink injection. A 50-µl blood sample was mixed with 4 ml of 0.1% sodium carbonate solution and the absorbance was determined at 660 nm.

The phagocytic index (K) was calculated using the following formula:



where, OD1 and OD2 are the optical densities at 0 and 15 min, respectively.[20]

Model for immunomodulation – cyclophosphamide induced neutropenia in mice

Animals were administered with study drugs as described in [Table 2] for 10 days. On the 10th day, neutropenic dose of cyclophosphamide (200 mg/kg, s.c.) was injected and the day marked as Day 0. Blood was collected from the animals through the retro-orbital plexus on a day prior to cyclophosphamide injection and on Day 3 after the injection. Estimation of total leukocytes count and differential leukocytes count was performed and the values were compared to determine the pharmacological effect of the herbal formulation.[20]

Statistical analysis

Statistical analysis was done using Graph Pad Prism version 4 software (Graph Pad Inc, USA). ANOVA followed by Dunnet’s multiple comparison tests was applied (all columns). Data was presented as Mean ± SEM. Confidence level was taken as 95%.


   Results Top


Model for pain – formalin-induced paw licking test in mice

In formalin-induced paw licking, Freemodex in high (160 mg/kg, p.o.), low (80 mg/kg, p.o.), and standard (diclofenac 10 mg/kg, p.o.) doses produced moderately significant reduction in paw licking, in both the phases, compared to control. The reported results were dose-dependent [Table 3].
Table 3: Effect of freemodex in formalin induced paw licking test in mice

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Model for inflammation – egg albumin induced hind paw edema in rats

In egg albumin induced paw edema, when Freemodex at a dose of 40 and 80 mg/kg (p.o.) and the standard (diclofenac 10 mg/kg, p.o.) was administered, a significant reduction in paw volume was observed compared with control after 5 h. The standard drug shows moderately significant decrease during the 1st, 2nd, 3rd, and 4th hours while the high dose of the test drug shows moderately significant reduction during the 1st and 2nd hours and significant reduction during the 3rd, 4th, and 5th hours [Table 2].

Model for arthritis – turpentine-induced arthritis in rats

In turpentine-induced arthritis, on all evaluatory days, administration of diclofenac sodium (10 mg/kg, p.o.) resulted in a moderately significant reduction in the diameter of the synovial joint when compared with control. The test herbal formulation Freemodex at a dose of 40 and 80 mg/kg, p.o. produced a moderately significant reduction in the diameter of the synovial joint when compared with control [Table 4].
Table 4: Effect of Freemodex in turpentine induced arthritis in rats

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Model for immunomodulation – carbon clearance test in mice

In the carbon clearance test, herbal formulation Freemodex at a dose of 80 mg/kg, p.o. produced a significant increase and at a dose of 160 mg/kg, p.o. produced a moderately significant increase in the clearance of the carbon particles from blood as indicated by significant increase in the phagocytic index [Table 5].
Table 5: Effect of Freemodex in carbon clearance test in mice

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Model for immunomodulation – cyclophosphamide induced neutropenia in mice

In cyclophosphamide-induced neutropenia, the test herbal formulation Freemodex, at a dose of 80 and 160 mg/kg, p.o., produced a moderately significant reduction in the total cell count and in the number of neutrophils [Table 6].
Table 6: Effect of Freemodex in Cyclophosphamide induced neutropenia in mice

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   Discussion Top


In recent times, polyherbal formulations have gained more importance because of their higher efficacy, easy availability and lesser side effects as compared to their synthetic counterparts. These herbal preparations are widely used by people for disease treatment and prevention as they display synergistic and potentiative action. It is believed that the mixture of different herbal species in these polyherbal formulations show better therapeutic effect than either of the species on its own.[21],[22],[23] Freemodex is a polyherbal formulation containing various medicinal plant ingredients [listed in [Table 7], marketed and manufactured by Jaffman Pharmaceuticals. Traditionally, it is used by locals for treatment of pain, especially, chronic joint pain. However, there is no preclinical data available to support this. Hence, the current study was designed to evaluate the analgesic, anti-arthritic, anti-inflammatory and immunomodulatory activity of the polyherbal formulation Freemodex against induced pain, inflammation, arthritis, and immunosuppression.
Table 7: Composition of Freemodex

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Formalin induced paw licking test in mice is a persistent-pain model, with two phases of pain and is widely used for determination of antinociceptive activity in rodents. In the first phase, bradykinins and tachykinins are released following direct activation of nociceptors and primary afferent fibres by formalin. Tissue injury occurs in the second phase, followed by release of histamine, prostaglandins, serotonin and other excitatory amino acids.[24],[25] NSAIDS antagonize the second phase while opioid analgesics inhibit both of them.[2],[26] Freemodex, in both low and high doses exhibit significant inhibition of both the phases of pain, indicating opioid analgesics-like action. This may be due to the presence of widely known analgesics Zingiber officinale,[27] Allium sativum,[28] Withania somnifera,[29] and Piper nigrum[30] in the polyherbal formulation.

Egg albumin induced hind paw edema is a nonspecific inflammation model frequently used to assess anti-inflammatory and antiedematous effects of natural products. Egg albumin induces release of inflammatory mediators: histamine and serotonin.[31] In this study, we found that high dose of Freemodex showed significant inhibition of the nonspecific inflammation. Evidence is significant reduction in paw volume after the 1st hour of egg white administration. It can inferred that the polyherbal formulation effectively inhibits release or action of the inflammatory mediators and especially possibly reduces the release of prostaglandins in the second accelerating phase of swelling as evident from the paw volumes of 4th & 5th hour.[32],[33],[34] Herbs like Piper nigrum,[30] Commiphora wightii,[35] Curcuma longa,[36] Asparagus racemosus,[37] Boswellia serrata,[38] and Terminalia chebula[39] present in Freemodex have proven protective effects against various initiators and mediators of inflammation like iNOS, COX-2, histamine, and prostaglandins.

In the present study, arthritis was induced by turpentine in rats to demonstrate the inhibitory effect of the polyherbal formulation Freemodex against Rheumatoid arthritis (RA). Freemodex when administered at doses 40 mg/kg, p.o. and 80 mg/kg, p.o. reported moderately significant reduction of paw volume and paw diameter. The reduction of paw volume and paw diameter in hind paw may be associated with inhibition of neutrophil infiltration, pannus formation and bone erosion. Inhibition of lipoxygenase and/or cyclooxygenase maybe also be one of the mechanisms behind the protective action of Freemodex.[3],[19],[33] Terminalia chebula, one of the herbs of the formulation is a potent inhibitor of iNOS and COX pathway.[39] Boswellia serrata,[38] Vitex negundo[40] and Phyllanthus emblica[41] present in Freemodex are known to possess protective effects against inflammation and arthritis.

Cyclophosphamide belongs to nitrogen mustard a subclass of alkylating agents which induces myelosuppression. The myelosuppression is caused by alkylation of DNA and interfering with DNA synthesis and function.[8],[42] The cyclophosphamide induced neutropenia model is to investigate the protective effects against the myelosuppression induced by the cyclophosphamide. Both doses of Freemodex 80 and 160 mg/kg, p.o. showed moderately significant prevention of neutropenia induced by the cyclophosphamide, may be through activation of the macrophages.[43]Carbon clearance test is done to evaluate the effects of drugs on the reticulo-endothelial system (RES). Phagocytes of the RES play a major part in clearance of particles from blood. This tendency of clearance of colloidal carbon particles by the macrophages from blood stream is termed as Phagocytic index and is measured using an exponential equation.[20] In this study, it is observed that Freemodex causes significant augmentation of the Phagocytic index. Such an effect may possibly be due to RES stimulation by certain herbs like Curcuma longa whose glycan fractions potentiate the RES[44] and Allium sativum that cause hypertrophy of the system.[45]


   Conclusion Top


This study demonstrates protective activity of the PHF − Freemodex − in experimental animal models. The results of this study suggest that the PHF is capable of alleviating distress due to pain and inflammation in animal models and may be used as an alternative to synthetic drugs. However, clinical studies are required to further prove its efficacy in humans.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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