INTRODUCTION:

Non-steroidal anti-inflammatory drugs (NSAIDs) are the mainstay for treatment and management of inflammation and pain. However, their use has always been accompanied with various adverse effects¹. Therefore despite the progress that has occurred in recent years in the development of therapy, there is still a space for better, effective drugs, especially for the treatment of chronic pain and inflammation. Recently discovered antinociceptive substances include secondary metabolites derived from plants. Plant-derived substances have significantly contributed the process of drug discovery, particularly in the development of new analgesic and antiinflammatory drugs². Cuscuta reflexa Roxb. (Dodder), (Convolvulaceae), is well known as Amarwel in Ayurveda. It is a parasitic climber found commonly throughout India. The plant has no root under the ground but only grows as a parasite twiner on other plants and hence called as ‘Akaswel’ (Sky twiner) or Amarwel(Immortal twiner), because it grows during the rains and every year the growth is fresh on the same plant³. From the dawn of history, Cuscuta reflexa has been used for various purposes viz. as a purgative, in the treatment of liver disorders, cough and itching, and for its carminative and anthelmintic actions⁴. The parasite is reported to possess preliminary antiinflammatory activity ^{5, 6}. In present study we tried to explore details antiinflammatory activity of extracts of Cuscuta reflexa using different models of acute as well as chronic inflammation. The emphasis was also given to find out mechanism involved in behind the antiinflammatory effect.

MATERIAL AND METHODS:

Plant material

Stems of Cuscuta reflexa growing on the plants of Ziziphus were collected from local region of Igatpuri, District Nasik in the month of February2011. Plant material was identified with the help of local community and was authenticated by Dr. P. G. Diwakar from Botanical Survey of India, Pune (Ref no. BSI/ WC/ Tech/ 2010/ 374).

Preparation of Extract and fractionalization

Plant material was separated from the host plant, cleaned, and dried under shade followed by pulverization. The coarse plant material was subjected to successive extraction method with solvent sequence of petroleum ether, methanol and water. Depending upon the biological activity and phytochemical analysis the methanol extract was fractionized in four fragments (Fr I-IV) using a silica gel column and a solvent system with ethyl acetate:formic acid:glacial acetic acid:water:methanol, 10.1:0.5:1.1:2.6:2 (v/v).

Phytochemical analysis and determination of phenolic content and flavonoid content of methanol and aqueous extracts.

The phytochemical analysis of the methanol and aqueous extracts of Cuscuta reflexa was done using standard methods⁷. Total phenolic content was estimated by the Folin Ciocalteu’s method and flavonoid content by aluminum chloride colorimetric assay, using gallic acid and rutin, respectively, as standards ^8,9 .

HPTLC fingerprinting and quantification of quercetin

HPTLC fingerprinting was performed on 20 cm × 10 cm aluminum backed plates coated with silica gel60F 254. Standard solution of quercetin and sample solution were applied to the plates as bands 8.0 mm wide, 30.0 mm apart, and 10.0 mm from the bottom edge of the same chromatographic plate by use of a Camag Linomat (Muttenz, Switzerland) sample applicator equipped with a 100-μL . Hamilton (USA) syringe. Ascending development to a distance of 80 mm was performed at room temperature (28 ± 2°C), with ethyl acetate:formic acid:glacial acetic acid:water:methanol, 10.1:0.5:1.1:2.6:2 (v/v), as mobile phase, in a Camag glass twin-trough chamber previously saturated with mobile phase vapors for 20 min. After development, plates were dried with a hair dryer and then scanned at 380 nm with a Camag TLC Scanner with winCATS software, using the deuterium lamp.

Animals

Albino (Wister) rats 180-200 g of either sex and albino mice (20-25 g) were used. The animals were kept in the standard polypropylene cages and provided with food and water ad libitum. The animals were acclimatized for a period of 14 days prior to performing the experiments. The experimental protocols were approved by Institutional Animal Ethics Committee (SMBTCOP-IAEC/ CPCSEA Reg. no. 1329/ac/10/CPCSEA).

Acute oral Toxicity study

Acute oral toxicity studies were performed according to OECD (2001)-425 guidelines (Limit test, Up and Down method). Swiss mice (n = 5) of either sex selected by random sampling technique were employed in this study. The animals were fasted for 4 h with free access to water only. The extracts were administered orally at a dose of 2000 mg/kg sequentially to the animals and mortality was observed for 3 days. Only one animal received test dose at a time.

Anti-nociceptive activity

Hot Plate method

Analgesic activity was tested in rats using the hot plate method of Janssen and Jagneau ^{10 .}Swiss albino mice of either sex who showed forepaw licking or jumped within 20 sec from hot plate kept at 55°C were selected for the experiment. Eligible animals were divided into eleven groups of six mice each and pre-treatment reaction time for each mouse was determined. Mice in the different groups were then treated with distilled water (10 ml/kg, p.o.), PECR (50,200 and 300 mg/kg, p.o.), MECR (50,200 and 300 mg/kg, p.o.), AECR (50,200 and 300 mg/kg, p.o.), and pentazocin (10 mg/kg,i.p.). Sixty minutes after oral and 30 min after intraperitoneal administration, the reaction time of animals was again recorded. Apost-treatment cut-off time of 20 s was used to prevent animal paw tissue damage ¹¹.

Acetic acid induced writhing

The writhing test in mice was carried out by using the method of Young¹². The writhes were induced by intraperitoneal injection of 1.0% acetic acid (v/v, 0.1 ml/10 g body weight). Drugs and control vehicles were administered 60 min before chemical stimulus. The number of muscular contractions was counted over a period of 5 min after acetic acid injection. The data represented the total numbers of writhes observed during 10 min.

Formalin Test

This test was based on the method of Lu et al. 2007¹³. 2% Formalin (50µl) was injected subcutaneously into the right hind paw of mice. The time (in seconds) spent in licking and biting responses of the injected paw was taken as an indicator of pain response. Responses were measured for 5 min after formalin injection (early phase) and 20-30 min after formalin injection (late phase). Drugs and control vehicles were administered 60 min before the formalin injection. DCS (10 mg/kg, p.o.) was used as standard and control animals received distilled water by oral route.

Anti-inflammatory activity

Carrageenan induced rat paw edema

Anti-inflammatory activity was evaluated using the Carrageenan induced rat paw edema according to the technique of Winter et al. (1962)¹⁴. After 16 h of fast the rats were divided into eleven groups of six animals each. Group I served as control group and received distilled water (DW), orally. Group III to XI animals received PECR, MECR and AECR (50, 200 and 300 mg/kg p.o.) respectively. Group II received DCS at a dose of 10 mg/kg p.o. After 1 h, 0.1 ml of 1% w/v carrageenan suspension was injected subcutaneously in to the plantar surface of the right hind paw. The paw volume was measured using a plethysmometer immediately and periodically up to 3 h after carrageenan injection.

Mediator induced rat paw edema

Different mediators of inflammation were used to induce edema in rat paws for the evaluation of probable mechanism of antiinflammatory activity; 0.1 ml solution of histamine base (1mg/ml), serotonin (1mg/ml) were injected into the right hind paw and the edema volume was determined immediately and after 60 and 30 minutes following histamine and serotonin injections, respectively¹⁵

Cotton pellet induced granuloma (Winter and Porter, 1957)

Autoclaved cotton pellets weighing 10 ± 1mg each were implanted subcutaneously through small incision made along the axilla or flank egion of the rats anesthetized with ether. Different groups of rats were administered the PECR, MECR and AECR (50, 200 and 300 mg/kg p.o.) and DCS (10 mg/kg, p.o.) once daily for 7 consecutive days from the day of cotton pellet insertion. The control group received vehicle (1 ml/kg, p.o.). On the eighth day, all the rats were sacrificed and the cotton pellets covered by the granulomatous tissue were excised and dried in hot air oven at 60 °C till a constant weight was achieved. Granuloma weight was obtained by subtracting the weight of cotton pellet on 0 day (before start of experiment) from the weight of the cotton pellet on eighth day.

Evaluation of mechanism of antiinflammatory activity

Antiulcerogenic activity

Adult Swiss albino rats (150-200 g) were fasted for 24 h. after the fasting period; MECR and AECR (300 mg/kg p.o.) were administered. Animals from standard group received DCS (10 mg/kg p.o.), and Control animals received distilled water. Three hours after drug administration, animals were sacrificed and stomachs were removed, cut along the lesser curvature and opened up to expose the mucosal surface. The mucosal surface was washed with normal saline and observed with a magnifying lens. Damage to the mucosa was scored 0 to 4 according to an arbitrary scale: 0 = no lesions; 0.5 = hyperemia; 1 = one or two lesions; 2 = severe lesions; 3 = very severe lesions; 4 = mucosa full of lesions. ¹⁶

Acetic acid-induced vascular permeability in mice

This test was followed by the method described by Whittle with some modifications¹⁷. Four groups of six mice per group were used for the study. Group I served as vehicle control, groups II was treated with DCS 10 mg/kg p.o., Gr. III and IV were treated with MECR and AECR (300 mg/kg p.o.), respectively. One hour after the treatment, 0.2 ml of 0.2% Evan’s blue in normal saline was injected intravenously through tail vein. Thirty minutes later, each mouse was injected intraperitoneally with 0.2 ml of 0.6% acetic acid in normal saline solution. After 1 h, the mice were sacrificed and the abdominal wall was cut to expose the entrails. The abdominal cavity was washed with 5ml of normal saline to collect pigments in a test tube. After centrifuging the contents of the tube to eliminate contaminants, the solution was subjected to colorimetric analysis using a spectrophotometer at a wavelength of 610 nm. The vascular permeability effects were expressed as the absorbance (A), which represented the total amount of dye leaked into the intraperitoneal cavity¹⁸ .

Leucocyte migration test

Adult Swiss albino rats (150-210 g) of either sex were used. The animals were randomly divided in to the groups of six animals and given the treatments. One hour after drug administration, animals received intraperitoneal injection of 1 ml of 6% w/v dextran in normal saline. Four hours later, the animals were killed and the peritoneal cavities washed with 5 ml of phosphate-buffered saline containing 0.5 ml of 10% EDTA. Total and differential leukocyte counts in the peritoneal wash were taken. The inhibition (%) of leukocyte migration was calculated¹⁹ .

Statistical Analysis

Each value represented the mean ±SEM of 3 consistent readings. The significance of the differences between controls and, tests were analyzed using analysis of variance followed by Dunnet multiple comparison test. Values of P<0.01are indicated by subscript ‘**’ and; Values of P<0.05 are indicated by subscript ‘*’ when compared with control.

RESULTS:

Phytochemical screening, Total phenolic, and flavonoid content of extracts of Cuscuta reflexa

Phytochemical investigation revealed the presence of saponins, sterols,alkaloids, flavonoids and glycosides in the extracts of Cuscuta reflexa. According to the present study, the total flavonoid content was determined to be 6.98 ± 0.40 mg rutin equivalents/gram extract; the total phenol content was 265.02 ± 4.70 mg gallic acid equivalents/gram extract.

HPTLC Analysis of extracts and characterization of bioactive fraction

HPTLC analysis of extracts of Cuscuta reflexa showed presence of flavonoids and confirmed presence of quercetin. MECR and AECR were found to contain 0.121% and 0.071% of quercetin respectively. Biological activity guided selection of fractions led to selection of Fr. II which according to the results of TLC and IR spectra is characterized as quercetin.

Acute oral Toxicity study

No toxic effects were observed in any animal, the LD 50% is more than 2000 mg/kg and the extracts can be regarded as safe.

Anti-nociceptive activity

Hot Plate method

PECR, MECR and AECR possess significant analgesic activity (P < 0.01).The results . indicate that oral administration of PECR, MECR, and AECR (200 and 300 mg/kg p.o.) inhibited thehot-plate analgesia and significantly increased the latency in treated animals.

Acetic acid induced writhing and Formalin Test

The effects of different extracts of Cuscuta reflexa on Acetic acid induced writhings shows that writhing response in untreated mice (41.33±0.71) was significantly reduced (P < 0.01) by MECR and AECR (at 200 and 300 mg/kg p.o.). Similarly formalin induced licking in an untreated mice (176.3±4.19) was reduced significantly (P < 0.01) by MECR and AECR (at 200 and 300 mg/kg p.o.).

Anti-inflammatory activity

Carrageenan and mediator induced rat paw edema

Results in Table 02, and fig. 03 show that MECR caused significant (P < 0.01) inhibition of paw edema induced by carrageenan and mediators like serotonin and histamine in dose dependent manner as compared to the control group.

Cotton pellet induced granuloma

Both MECR and AECR found to inhibit the cotton pellet induced granuloma formation significantly (P < 0.01) at doses 200 mg/kg p.o. and 300 mg/kg p.o., whereas PECR also inhibit the granuloma significantly(P < 0.01) only at higher doses (300 mg/kg p.o.)

Evaluation of mechanism of antiinflammatory activity

Antiulcerogenic activity

Rats treated with diclofenac sodium were found to have severe gastric lesions with the score 3.17±0.31. Whereas the animals treated with MECR and AECR (300 mg/kg p.o.) were having only few lesions with the scores 1.50±0.22 and 1.75±0.36, respectively

Acetic acid induced vascular permeability

Oral administration of 300mg/kg of MECR and AECR evoked a significant (P < 0.01) inhibition of vascular permeability (56.25±2.39% and 49.20±2.93%, respectively) induced by acetic acid in mice. DCS inhibits the permeability by 69.88±2.39 %

Leukocyte migration test

The total leukocyte count was significantly inhibited in animals treated with AECR and MECR (300 mg/kg p.o.)i.e. 50.65±2.74% and 48.36±3.20 %, respectively. DCS inhibits the migration by 73.98±0.84 %

DISCUSSION:

Results of acute toxicity test revealed that various extracts are safe and tolerable with no obvious adverse effects till the dose level of 2 g/kg of body weight. Phytochemical investigations revealed presence of various secondary metabolites in extracts of Cuscuta reflexa, HPTLC analysis confirmed the presence of flavonoids like quercetin. Depending upon biological activity, MECR was selected for separation and was fractionized in to four (Fr I-IV) fractions. IR spectra of fraction II of methanol extract confirmed the presence of quercetin.

The anti-nociceptive and anti-inflammatory effects of AECR and MECR were investigated in in vivoanimal models. Anti-nociceptive activity was evaluated using various animal models like Hot plate analgesia; acetic acid induced writhing response and formalin test. Hot plate analgesia is a sensitive acute pain test for detecting opiate analgesia as well as several types of hyperalgesic reactions from spinal origin²⁰. The results indicate involvement of central mechanism. In another study, antinociceptive effect was evaluated using acetic acid induced writhing test. Writhing responses are characterized by abdominal contractions followed by extension of hind limbs, induced by intraperitoneal injection of acetic acid. The writhing test is simple and sensitive tool for rapid evaluation of mild analgesic non-steroidal antiinflammatory drugs. Intraperitoneal administration of acetic acid causes release of inflammatory mediators like cyclooxygenase(COX), lipoxygenase (LOX), prostaglandins(PGs), histamine, serotonin, bradykinin, substance P, IL-1b, IL-8, TNF-α in the peripheral tissue fluid. Increased level of these mediators causes the sensitization of primary afferent nociceptors entering dorsal horn of the central nervous system. These mechanisms are responsible for the development of inflammatory pain and abdominal constriction and this effect is supposed to be a peripheral pathway^21.Results indicate significant inhibition of writhings by pretreatment of animals with AECR and MECR suggesting a peripherally mediated analgesic activity based on inhibition of the stimulation of peripheral receptors especially the local peritoneal receptors at the surface of cells lining the peritoneal cavity, an effect related with inhibition of mediators of inflammation. As the results of two tests for nociception indicated two different mechanisms, Formalin test was used for detailed study of mechanism of nociception. The formalin test produces a distinct biphasic response and different analgesics may act differentially in the early and late phases of this test and hence the test can be used to clarify the possible mechanism of antinociceptive effect of a proposed analgesic ²². Early phase is characterized by neurogenic pain, which is induced by direct chemical stimulation of the nociceptors, particularly C-fibers. The involvement of substance P and bradykinin has also been reported. Centrally acting drugs like opioids inhibit both phases equally. In the late phase on the other hand, inflammatory pain is induced by production and action of different inflammatory mediators like prostaglandins (PGs), histamine, bradykinin, and serotonin in peripheral tissues ²³. The effect of AECR and MECR on the late phase of formalin test suggests that its activity may be resulting from its peripheral action which may be related with the inhibition of inflammatory mediators, inhibition of thermally induced pain could be the result of presence of such antiinflammatory substances in Cuscuta reflexa.

Antiinflammatory activity of MECR and AECR was estimated using various animal models, namely Carrageenan induced rat paw edema, mediators induced rat paw edema, which represents acute phase of inflammation, and Cotton pellet induced granuloma.

Carrageenan-induced inflammation consists of three distinct phases, first phase of which is characterized by release of histamine and serotonin; followed by a second phase mediated by kinins; and a third phase involving prostaglandins. Result of the present study indicates that AECR and MECR significantly inhibit paw edema. Inhibition of paw edema in the first phase may be due to inhibition of the release of early mediators, such as histamine and serotonin, and the suppression in the second phase may be because of an inhibition of kinins or prostaglandins²⁴. For better understanding of underlying mechanism of antiinflammatory effect of AECR and MECR, their effect on mediators like serotonin and histamine was evaluated and result of mediator induced inflammation indicates significant inhibition of both histamine and serotonin induced paw edema by AECR and MECR suggesting inhibition of release or action of histamine and serotonin.

Acute inflammation may lead to chronic inflammation if the injurious agent persists or the process of healing is interrupted. Effects of MECR and AECR on chronic inflammation were evaluated by using cotton pellet granuloma. Granuloma formation in chronic inflammation is characterized by proliferation of macrophages, neutrophils, fibroblasts and multiplication of small blood vessels, giving rise to formation of a highly vascularised reddish mass, i.e. granuloma ²⁴. NSAIDs are found to inhibit granuloma formation by virtue of preventing granulocyte infiltration to the foreign body implanted²⁶. Result of the present study indicates significant inhibition of granuloma formation by both MECR and AECR.

To understand the probable mechanism involved in this effect, various methods were employed. Gastric ulcerogenicity test was performed according to the method of Cashin²⁵ . Gastrointestinal side effects are commonly encountered adverse effects associated with orally ingestedanti-inflammatory or anti-arthritic agents, the risk of gastrointestinal ulceration, bleeding and even perforation with these drugs. The mechanisms behind gastro-intestinal irritation are complex. Deleterious effects may result from local actions, which cause injury to the local blood vessel or may involve inhibition cyclooxigenase (COX) which results in inhibition of synthesis of protective prostaglandins which is seen in case of nonspecific (COX) inhibitors. Macroscopic examination of the gastric mucosa of rats treated with MECR and AECR did not produce severe lesions which are seen with standard NSAIDs indicates therapeutic advantage over conventional drugs and indicates selective inhibition of COX2 which is an inducible enzyme at the sites of inflammation^25.

Acetic acid-induced vascular permeability causes an immediate sustained reaction that is prolonged over 24 h and its inhibition by MECR and AECR suggests that the extract may effectively suppress the exudative phase of acute inflammation.

Leukocytes migration induced by dextran, was used to analyze the effect of MECR and AECR on leukocyte migration. Dextran induced leukocyte migration is known to cause the accumulation of edema fluid containing proteins and leukocytes. Result of the study indicate significant (P<0.01) inhibition of leukocyte migration. The concentration of neutrophils at peritoneal cavity was also significantly (P<0.01) low as compared to control animals. The potent antiinflammatory and analgesic effects produced by extracts of Cuscuta may be attributed to the secondary metabolites like quercetin present in the plant.

CONCLUSION:

This study demonstrated that MECR and AECR exhibited significant analgesic effect against nociceptive stimulus generated by hot plate method, acetic acid (i.p.), and formalin (intraplantar) injection and antiinflammatory activity against acute inflammatory responses like, carrageenan, histamine and serotonin induced paw edema. The extracts were equally effective in proliferative as well as chronic inflammatory reactions as evident from results of cotton pellet granuloma. The anti- inflammatory mechanisms of MECR and AECR are considered closely related to inhibition of release of inflammatory mediators like serotonin and histamine, COX inhibition (and hence prostaglandin synthesis), inhibition of Vascular permeability as well as inhibition of neutrophil infiltration. Thus it can be said that extracts of Cuscuta reflexa possess complex mechanism of antiinflammatory effect. The anti-inflammatory and analgesic effects of extracts are correlated and share common molecular pathways, and can be attributed to the presence of quercetin. By virtue of its effects it can be developed as a therapeutic agent for the treatment of various inflammatory diseases.

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Received on 05.04.2014 Modified on 25.04.2014

Res. J. Pharmacology & P’dynamics. 6(2): April- June 2014; Page 112-117