INTRODUCTION:

Inflammation is a non-specific defense response of the body to the tissue damage. Among the conditions that may produce inflammation are the pathogens, abrasions, chemical irritations, distortions, or disturbances of the cells, and extreme temperatures. The four characteristic signs and symptoms of inflammation are redness, pain, heat, and swelling. Inflammation can also cause a loss of function in the injured area, depending on the site and extent of the injury. Inflammation is an attempt to dispose of microbes, toxins, or foreign material at the site of injury, to prevent their spread to other tissues, and to prepare the site for tissue repair in an attempt to restore tissue homeostasis¹.

Inflammation can result in locally increased production of free radicals by inflammatory enzymes, as well as the release of inflammatory mediators that promote cell proliferation and angiogenesis and inhibit apoptosis. Although the currently used steroidal and non-steroidal anti-inflammatory drugs treat acute inflammatory disorders, these conventional drugs have not been successful in curing chronic inflammatory disorders such as rheumatoid arthritis.

Moreover, usage of many of such drugs has been associated with adverse impact upon the hepatic and renal functions^{2- 4}.

Pain or algesia is an unpleasant and emotional experience caused by real or potential injury or damage to the body. Scientist believes that pain evolved in the animal kingdom as a valuable three part warning system. First, it warns of injury, second, it protects against further injury causing reflexive withdrawal from the source of injury. Finally, it leads to a period of reduced activity, enabling injuries to heal more efficiently. Pain is difficult to measure in humans because it has emotional, psychological component as well as physical component. Some people express extreme discomfort from relatively small injuries, while others show little or no pain even after suffering severe injuries. Sometimes pain is present even though no injury is apparent at all, or pain lingers long after an injury appears to have healed⁵.

Literature survey revealed that the plant Commicarpus chinensis has been used in various ayurvedic and siddha preparations as hepatoprotective, gastroprotective, in Down’s syndrome, analgesic, antipyretic, and anti-inflammatory agent⁶.

Table.1: Analgesic effect of C.chinensis in Acetic acid induced writhing

Treatment Groups	Average No. of writhings / 15 mins	% Inhibition
Control	49.17±0.47	------
Standard	19.17±0.30	61.01
Aq. Extract 1	29.67±0.42***	39.65
Aq. Extract 2	24.50±0.42***	50.17

Values are the Mean± S.E.M, n=6, Significant *P <0.05 compared with Standard

Recently the use of herbal medicines has been increased all over the world due to their miraculous therapeutic effects and fewer adverse effects as compared to the modern medicines. Presently available categories of drugs like NSAIDS have been implicated to have many side effects like hyperacidity, ulcerations while those belonging morphine and its analogues have adverse effects ranging from CNS depression to physical dependence, abuse liability associated with the use of CNS acting analgesics, antipyretics and anti-inflammatory agents⁷.

Commicarpus chinensis is a perennial herb, commonly known as gum fruit belonging to the family Nyctaginaceae⁶. The other synonyms for the plant are Boerhavia chinensis, Boerhavia repanda, and Valerian chinensis⁸. It is commonly known as diffuse hogweed. In Marathi, it is known as Punarnava, Pungli and in Telugu, it is called as Theega mamidi⁸. Many parts of C. chinensis plant has been included in various treatments, like the roots are used for edible purpose for its nutritional properties⁹ and used in treatment of skin diseases¹⁰. The plant has got diuretic activity¹¹. It has antioxidant activity and free radical scavenging activity⁶. Traditionally the Roots are taken orally for its antihelmintic, leucorrhea, and Galactoseamine and paracetamol intoxication effects¹⁰. Phytochemical investigation of the plant revealed that the plant contains carbohydrates, monosaccharide, amino acids, steroids, saponins glycosides, tannins, phenolic compounds, flavonoids⁶.

Among the prescription drugs, analgesics and anti-inflammatory drugs are the foremost medications in terms of frequency of use. Every day these are used by more than 30 million individuals globally; of these 40% of the consumers are older than 60 years. Only 4.5% of the prescriptions are for so-called centrally acting analgesics i.e. opioids. Population studies have shown that 10-20% of all the people who are 65 years or older either are receiving or have received a prescription for NSAIDS. During the next 20 years, the number of people over 65 is expected to increase from 380 million to 600million. The very frequent use of NSAIDS is based on the fact that these agents have many indications for which a large number of patients exist. These indications include chronic polyarthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, gout, inflammatory soft tissue rheumatism, low back pain, post operative and post traumatic inflammation, thrombophlebtits and vasculitis¹².

The major disadvantage with the use of presently available potent synthetic drugs is their associated side effects like nausea, vomiting, constipation, ulcers, respiratory depression, hypotension, toxicity and reappearance of symptoms after discontinuation. Hence the search for new anti-inflammatory and analgesic agents which can retain the therapeutic efficacy and yet devoid of adverse effects are justifiable.

MATERIAL AND METHODS:

Animals:

Wistar albino rats (180-200g) and Swiss albino mice (18-22g) of either sex were used in the experiment. The animals were allowed to acclimatized to the laboratory conditions for 07 days and housed under standard conditions of temperature (25±2ºC) and relative humidity (30-70%) with a 12h light-dark cycle and were fed with standard pellet diet (Hindustan liver co., Mumbai) and water ad libitum. The animal studies were performed in accordance to guidelines of CPCSEA.

Plant Material:

The plant C. chinensis was procured from the campus of Gulbarga University, Gulbarga and authenticated by the Department of Botany, Gulbarga University, Gulbarga, Karnataka. Care was taken to collect the plants which were free from diseases and pest. After the collection and authentification, the plant material was washed under running tap water to remove adhering dust. The plants were air dried under shade and powdered. The resulting powder material was subjected to extraction with water.

Preparation of Extracts:

The plant material is shade dried and coarsely powdered. Coarsely powdered material was subjected to extraction using soxhlet apparatus. Distilled water was used as a solvent. The powdered material was evenly packed in extractor for 36h with distilled water. The temperature of the electric heating mantle was maintained using thermostat control. Appearance of colorless solvent in the siphon tube is considered the end of extraction process. The extract was concentrated by distillation process. The extract was air dried at room temperature and weighed.

Acetic Acid Induced Writhing Test¹³:

Albino mice (18-22g) were fasted for 12hr before starting experiment. Animals were divided into 04 groups. Group I was injected with normal saline (10 ml/kg; p.o.) as served as control. Group II was injected with standard drug aspirin (100mg/kg; p.o.) and the remaining 02 groups were administered with 100 and 200 mg/kg; p.o. of extract. After 30 min of saline, standard and extracts (CC1 & CC2) administration, the animals were treated i.p., with 1% acetic acid. The writhing was counted after 5 min of acetic acid injection. The number of abdominal constrictions (writhing) was counted for 15 min. The percent analgesia was calculated using the following formula:

Percent analgesia =

No. of writhing in Control

No. of writhing in test

X 100

No. of writhing in control animals

Carrageenan Induced Paw Edema in Rats¹³:

During anti-inflammatory studies, paw edema was induced by injecting 0.1 ml of 1% (w/v) carrageenan suspension into the sub planter region of the right hind paw of the rats (Mahat and Patil, 2007). The control group - I was orally administered saline (10 ml/kg) while the standard group - II was given indomethacin (5 mg/kg), and groups CC1 and CC2 were given 100 and 200 mg/kg of the aqueous extract 1h before carrageenan injection. The measurement of paw edema was carried out by displacement technique using plethysmometer to find out the circumference of paw edema immediately before and after 1, 2, 3 and 4 h following the carrageenan injection. Percentage inhibition is calculated with the following formula –

(Ct - Co) control – (Ct - Co) treated

% Inhibition =________________________________× 100

(Ct - Co) control

Statistical Analysis:

The results were articulated as mean± SEM of six animals. For statistical analysis, one way ANOVA was followed by Dunnett’s test for multiple comparisons were used. Effects were considered to be significant at P < 0.05 level.

RESULTS:

Analgesic activity (acetic acid induced writhing analysis)

The analgesic effect of the extracts was evaluated based on the number of writhings produced following the treatment of the animals with the drugs. The reduction in the writhing up to 15min was significantly higher (p < 0.05) at the dose of 100mg/kg (39.6%) as well as at 200mg/kg (50.1%) when compared with the standard group (61%) (Table.1). Comparing the analgesia of the test drug with the standard drug, the observed effect was slightly less at the test drug dose of 200 mg/kg as indicated by 50.1% inhibition in writhings as compared to the standard drug aspirin which produced 61% inhibition.

Anti-inflammatory studies (carrageenan-induced paw edema):

The circumference of paw edema in rats induced by the carrageenan was used to assess acute anti-inflammatory effect of C.chinensis at a dose of 100 and 200mg/kg dose. It showed significant decrease in inflammation. As indicated by the percentage decrease of 72.5% and 74.8% respectively up to 4h as compared to the standard group with percentage protection 79.1% (Table.2). The percentage inhibition of the acute inflammation in the paw of rats was found to be less effective in a dose of 100mg/kg when compared to 200mg/kg and standard respectively.

DISCUSSION:

Pain remains a high unmet medical need and a significant number of patients are not effectively treated with currently available therapies. There is a significant challenge in developing more effective therapies to treat pain, particularly in chronic debilitating pain conditions such as neuropathic pain.¹⁴

Inflammation is a complex biological response of the vascular tissues to harmful stimuli including pathogens, irritants, or damaged cells. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. The process of inflammation is necessary in healing of wounds. Inflammation however, if runs unchecked lead to onset of disease like vasomotor rhinnorhoea, rheumatoid arthritis and atherosclerosis. Acute inflammation is characterized by the classical signs – edema, erythema, pain, heat and above all loss of function. These signs are triggered by the infiltration of the tissues by serum and white blood corpuscles. Chronic inflammation results in a progressive shift in the type of cells present at the site of inflammation. It is characterized by simultaneous destruction and healing of the injured tissue from incidence of inflammation.¹⁵

On the basis of the results obtained from the preliminary Phytochemical investigations and in vitro antioxidant studies, it is confirmed that the aqueous extract of Commicarpus chinensis contains large amounts of flavonoids, saponins and phenolic compounds and exhibits high antioxidant and free radical scavenging activities. It also scavenges NO and DPPH free radicals and has reducing power. These in vitro assays indicate that this plant extract is a significant source of natural antioxidant, which might be helpful in preventing the progress of various oxidative stresses.⁶

The aim of the present study was to investigate the anti-inflammatory and analgesic effects of the aqueous extract of C.chinensis on acute inflammation induced by carrageenan induced paw edema and peripheral analgesia produced by acetic acid induced writhing methods at a dose of 100 and 200mg/kg; p.o.

The evaluation of peripheral analgesic effect of the test drug exhibited significant percentage inhibition in the abdominal writhings which were induced by acetic acid in the mice at both the 100 and 200mg/kg doses of the aqueous extracts when compared with the standard drug (Table. 01). The percentage inhibition of writhings at the higher dose of 200mg/kg indicated the pronounced peripheral analgesic effect in visceral pain which was comparable to the pure drug morphine within 15 min of test. The significant analgesic effect at the higher dose was due to the presence of high concentration of flavonoids which inhibited the synthesis, release or activation of receptor mediated responses by prostaglandins.

The assessment of acute inflammation which was induced by the carrageenan in the paw of rats demonstrated the significant percentage decrease in volume of paw edema following administration of the aqueous extract of C.chinensis in doses of 100 and 200mg/kg; p.o. The percentage inhibition of acute inflammation in the paw of rats was found to be lower in case of 200mg/kg of test drug dosage when compared to the standard drug indomethacin after 4h of treatment (Table. 02).

The acute and sustained effect on the inflammation up to 4h depends upon the reduced production of pre-inflammatory cytokines and PGE₂ in the tissues of the effected part of the body. The inflammation induced by phlogostic agent is related to the production of histamine, bradykinin and cyclooxygenase products while delayed phase is related to neutrophil infiltration, as well as to the continuing of the production of arachidonic acid metabolites. Prostaglandins and nitric oxide biosynthesis is involved in inflammation, and isoforms of inducible nitric oxide synthase (iNOS) and of cyclooxygenase (COX-2) are responsible for the production of a great amount of these mediators. It has been demonstrated that flavonoids are able to inhibit both enzymes, as well as other mediators of the inflammatory process such as reactive C protein or adhesion molecules.¹⁶

The variety of flavonoids have been found to have antimicrobial, antiviral, anti-ulcerogenic, cytotoxic, anti-neoplastic, mutagenic, antioxidant, antihepatotoxic, antihypertensive, hypolipidemic, antiplatelet and antipyretic anti-inflammatory activities. Flavonoids also have biochemical effects, which inhibit a number of enzymes such as aldose, reductase, xanthine oxidase, phosphodiesterase, Ca⁺²-ATPase, lipoxygenase, cyclooxygenase, etc.^17-19

The detailed scientific evaluation of the pharmacological properties of the aqueous extract of the test drug in terms of analgesic and anti-inflammatory actions clearly indicates its significant therapeutic efficacy which was found to be comparable to that of the standard drugs. The test drug was significantly effective and has sustained effect along with no indications of acute toxicity in the preclinical study. The analgesic and anti-inflammatory effects are inter-related and these effects are due to the presence of flavonoids in the aqueous extract.

CONCLUSION:

From the results obtained, it can be concluded that the aqueous leaf extract of C.chinensis poses significant anti-inflammatory and analgesic activity. As the aqueous extract in doses 100mg/kg and 200mg/kg significantly reduced the volume of paw edema in rats and writhing in mice. The effectiveness of the extracts is due to the presence of flavonoids, saponins and tannins which are known to have antioxidant properties and the efficacy was found to be dose dependent.

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Received on 16.04.2014 Accepted on 12.05.2014

Asian J. Pharm. Tech. 2014; Vol. 4: Issue 2, Pg 59-62

Treatment Groups	Volume of edema / Time					% Inhibition
Treatment Groups	0h	1h	2h	3h	4h	% Inhibition
Control	0.480±0.00	1.433±0.03	2.522±0.04	2.805±0.01	2.930±0.02	-----
Standard	0.466±0.02	0.880±0.01	0.765±0.06	0.666±0.03	0.611±0.03	79.14
Aq. Extract 1	0.436±0.01	0.953±0.04***	0.883±0.04**	0.836±0.03***	0.805±0.04***	72.52
Aq. Extract 2	0.455±0.00	0.983±0.03**	0.860±0.05*	0.808±0.05***	0.736±0.03***	74.88