Research Paper Summary: Analgesic and Anti-inflammatory Activities of Polyrhachis

Analgesic and Anti-inflammatory Activities of Total Extract and Individual Reactions of Chinese Medicinal Ants Polyrhachis Lemellidens

Junping Kou, Yun Ni, Na Li, Jinrong Wang, Liang Liu and Zhi-Hing Jiang, published in The Biological and Pharmaceutical Bulletin of the Pharmaceutical Society of Japan, 2005. Original paper available here. What follows is our summary.

The ethanol extract of Chinese medicinal ants Polyrhachis lamellidens was evaluated for its analgesic and anti-inflammatory activities in mice. It was shown that the extract significantly inhibited acetic acid-induced writhing response and increased hot-plate pain threshold of mice at doses of 1.5 and 3.0 g/kg. Such action was similar to that of the ethanol extract of Polyrhachis vicina Roger, which showed significant analgesic effect after oral administration of doses at 5.62 and 11.25g/kg.

Meanwhile, the extract significantly inhibited the increase in vascular permeability in acetic acid-induced peritoneal cavity irritation and in xylene-induced ear edema in mice. However it did not significantly inhibit leukocyte migration. These results suggest that P. lamellidens presents remarkable analgesic and anti-inflammatory activity, which supports the traditional use of medicinal ants of genus Polyrhachis in the treatment of various diseases associated with inflammation, including rheumatoid arthritis and chronic hepatitis.

Ants (Family Formicidae) are commonly and widely distributed all over the world. Although they are not commonly included in the diet of developed countries, they do in fact contain a rich array of nutritional components including vitamins, proteins, amino acids and trace elements such as zinc, selenium and manganese. In addition, ants have been used as medicine, owing to their special active substances such as citral (C10H16O), citionella terpene, ATP, histamine, growth hormone, testosterone and superoxide dismutase (SOD).

Of the many kinds of ants that have been used (e.g., Polyrhachis vicina Roger, Polyrhachis lamellidens Smith, Formica Fusca L., Formica Sanguinea L., etc.), the black ant, Polyrhachis vicina Roger has been extensively studied, showing that it possesses many pharmacological properties, such as reducing inflammation, relieving pain, retarding aging, reducing stress, reversing tumor growth, protecting liver, decreasing blood glucose, modulating immunological function and enhancing gonadal function, and has been used and has been used to treat various diseases such as arthritis and hepatitis in China for many years.

Less is known about the pharmacological activities of other kinds of medicinal ants. Another species in the Polyrhachis genus, P. lamellidens Smith, which is widely distributed in the mainland of China, Hong Kong, Taiwan, Japan and Korea, has also been used for the treatment of inflammatory diseases including arthritis and hepatitis for centuries in China, and is the subject of this study. We previously undertook chemical studies on the constituents of P. lamellidens and have isolated and structurally identified a disaccharide trehalose and two novel aliphatic polyketide lactones from its ethanol extract.

Acetic Acid induced Writhing Response in Mice

The test was carried out using Koster’s technique. Mice were injected intraperitoneally with acetic acid solution in saline, then monitored for writhing events indicative of pain. Prior to the injection, Polyrhachis extract was given to one group of mice and Indomethacin [A powerful NSAID and Cox-1 and COX-2 inhibitor associated with severe side effects -Ed.] was administrated to another group of mice in this experiment for comparison, while another group of mice received no medication.

Ethanol extract of Polyrhachis lamellidens inhibited the writhing response with inhibition percentage of 62.3% and 75.9%, depending on dosage. The control drug, Indomethacin, also significantly inhibited the writhing response at an inhibition percentage of 82.6%. Meanwhile during the experimental process, mice given the polyrhachis extract were not observed to show any obvious other behavioral changes (including excitation, sedation, muscle relaxant etc.).

Hot Plate Latency Assay in Mice

Using the method described in Eddy et al., mice were placed on hot plate and monitored for onset of pain response, e.g., shaking or licking hind paws or jumping. Mice with baseline latencies of 10 or 30 seconds were eliminated from the study. A manual 60 second cut-off was used to prevent tissue damage. Prior to hot plate tests, Polyrhachis extract was given to one group of mice, Rotundine was administrated to a second group of mice for comparison, while a control group received no medication.

Extract of Polyrhachis significantly increased hot-plate latency [delay of pain response - Ed.] of mice at doses of 1.5 and 3.0 g/kg, with a peak at 1 hour after drug administration. Results were nearly identical to those achieved with Rotundine (10 mg/kg), which also markedly increased hot-plate latency of mice. During the experimental process, mice given extract of Polyrhachis were not observed to show any other behavioral changes.

Xylene-Induced Ear Edema in Mice

The xylene-induced ear edema test was performed as described in Dai et al.  Polyrhachis extract was given to one group of mice and Indomethacin was administrated to another group of mice in this experiment for comparison.

Each animal then received 30m l of xylene on the anterior and posterior surfaces of the right ear lobe. The left ear was considered as control. One hour later, the ear tissue was examined for edema [swelling indicative of inflammation - Ed.].

The ethanol extract of Polyrhachis at the dose of 3.0 g/kg remarkably inhibited xylene-induced swelling in mice with the inhibition percentage comparable to that of Indomethacin 10 mg/kg [See Fig. 3].

Polyrhachis exhibits anti-inflammatory effects in animal studies

 

 

Effects on Acetic Acid-Induced Peritoneal Capillary Permeability in Mice

Acetic acid can induce pain as well as cause increase of vascular permeability, hence it is often employed to evaluate the activities of anti-inflammatory drugs.

According to a modified method of Whittle, the mice were injected intravenously with Evans blue dye solution, immediately followed by an intraperitoneal injection of 0.1 ml/10 g body weight of 0.7% acetic acid. Then, peritoneal fluids were collected and vascular permeability was expressed in terms of dye (m g/mouse), which leaked into the peritoneal cavity according to the standard curve of Evans blue. 

Polyrhachis extract was given to one group of mice and Indomethacin was administrated to another group of mice in this experiment for comparison, while another group of mice received no medication.

The ethanol extract of Polyrhachis produced an remarkable inhibitory effect on peritoneal capillary permeability produced by acetic acid at a dosage of 3.0 g/kg (Fig. with results comparable to those of indomethacin. [See Fig. 4]

Polyrhachis exhibits anti-inflammatory effect in peritoneal permeability study

 

Leukocyte Emigration Induced by CMC-Na

Since leukocytes emigration participates in the inflammatory process, CMC-Na-induced leukocytes emigration tests are used to study anti-inflammatory action. Groups of mice were treated with Polyrhachis extract, Prednisone, and a control group was not treated with any medication.

The test was carried out using the technique of Qin et al.  Mice were injected intraperitoneally with 0.25 ml/kg body weight of 1% CMC-Na solution (250 mg/kg) in normal saline. Four hours after the injection, peritoneal fluid was examined and the number of leukocytes was counted under a light microscope.

The ethanol extract of Polyrhachis at doses of 1.5 and 3.0 g/kg orally, showed no inhibition on leukocyte emigrations induced by CMC-Na, while Prednisone as a positive-control remarkably decreased leukocyte counts in the mice peritoneal cavity of mice.

Conclusions For Analgesic Effects

The analgesic activities were evaluated by two animal models, The hot-plate test and the Acetic acid-induced peritoneal pain model.

Polyrhachis extract can significantly inhibit the pain response to hot plate stimulus at doses of 1.5 and 3.0 g/kg, while the comparison drug, Rotundine also exerted similar remarkable analgesic activity. It has been reported that Rotundine’s activity may be associated with b -endorphin neurons in the arcute nucleus and supraspinal D2 receptor. Therefore, we concluded that the ethanol extract of Polyrhachis lamellidens has a remarkable inhibitory activity in non-inflammatory reactions as well as inflammatory pain, and this activity may be related with suppression of synthesis and/or release of those endogenous proinflammatory substances.

Our results also show that the ethanol extract inhibited acetic acid-induced writhing in response to peritoneal pain in mice after oral administration for 1 and 2 h at doses of 1.5 and 3.0 g/kg, while the comparison drug, Indomethacin also showed similar remarkable inhibitory activity.

Our data regarding behavior of the various fractionated components of Polyrhachis extract suggests that the analgesic components of P. lamellidens exist mainly in nonpolar fraction, indicating the polyketide lactones isolated from the diethyl ether fraction may possess analgesic activity.

Conclusions For Anti-inflammatory Effects

Three animal models were chosen for the screening of anti-inflammatory effects.

Xylene-induced neurogenous edema partially associated with substance P was selected because of its convenience for handling. Acetic acid can induce pain as well as increase of vascular permeability, hence it is often employed to evaluate the activities of anti-inflammatory drugs.

At the same time, since leukocytes emigration participates in the inflammatory process, CMC-Na-induced leukocytes emigration was employed for the anti-inflammation assays in this study.

The results show that the ethanol extract of Polyrhachis lamellidens remarkably inhibited xylene-induced ear swelling and acetic acid-induced peritoneal capillary permeability in mice. However it has no obvious effect on leukocytes emigration.

This suggests that the ethanol extract of P. lamellidens exerts significant anti-inflammatory activity, especially in the acute phase of inflammation, but it can not affect leukocytes function.

The pharmacological data of the four fractions indicated that only the water fraction has remarkable anti-inflammatory effect in two animal models, suggesting that the anti-inflammatory components of Polyrhachis may exist mainly in the water fraction of the ethanol extract.