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The present study evaluated the anti inflammatory activity of ethyl acetate and aqueous extracts of bark of M. nagi using carrageenan and histamine induced rat paw edema. Adult Wistar albino rats were subjected to carrageenan and histamine induced rat paw edema tests. In carrageenan induced rat paw edema the effects of ethyl acetate and aqueous extract at 100 and 200 mg/kg showed % inhibition of edema 27% and 22% respectively than the standard drug aspirin (28%). These ethyl acetate and aqueous extract extracts also showed % inhibition of edema 25% and 18% respectively than the standard drug (27%) when rats challenged with histamine induced rat paw edema. Future research should focus on the identification and the anti inflammatory activity of the constituents from this plant.
Myrica nag Hook (syn. Myrica esculenta Buch. and Ham) (Myricaceae) is a subtropical shrub commonly known as Box berry. The medicinal uses and chemical constituents of M. nagi have been widely studied7. The constituents of M. nagi have been shown to inhibit toxicity in a number of animal model systems2,13. A number of the chemical constituents of M. nagi have been identified as strong antioxidants 7, and a number of pharmacological effects of M. nagi have been reported2,8,13. It has been traditionally used for the treatment of various disorders such as liver diseases, fever, asthma, anemia, chronic dysentery, ulcer and inflammation9,13. M. nagi bark contains gallic acid, myricanol, myricanone, epigallocatechin 3-O-gallate, two prodelphinidin dimers [epigallocatechin-(4β→8)-epigallocatechin 3-O-gallate and 3-O-galloyl epigallocatechin-(4β→8)-epigallocatechin 3-O-gallate], and the hydrolyzable tannin castalagin14.
Inflammation is a tissue reaction to infection, irritation to foreign substance and form an integral part of host defense mechanisms. Inflammation which was recognized as a simple allergic reaction for many decades12. There are four cardinal signs of inflammatory condition and they are redness, heat, swelling and pain.
There are several tissue factors or mechanisms that are known to implicate in the inflammatory reaction such as histamine, bradykinin and prostaglandins4 and inflammation is a homeostatic phenomenon. The present work was down to screen anti inflammatory activity of the plants to throw more light in this direction.
Barks of M. nagi (MN) were purchased from a local market. The plant was identified and authenticated by S. Kitchlu, Indian institute of integrative medicine (CSIR), Jammu, India. A voucher specimen (SU/DPS/Herb/32) of the same has been deposited in the Department of Pharmaceutical Sciences, Saurashtra University, Rajkot for future reference.
Barks were dried in shade, moderately ground by electric grinder and subjected to soxhlet extraction using ethyl acetate and later solvent was evaporated at reduced pressure to afford ethyl acetate extract (yield-5.2% w/w). M. nagi aqueous extract was obtained by boiling fresh powder in distilled water (100° C) and later by evaporating water from the decanted portion under reduced pressure (yield-29.4% w/w). The extracts were stored in refrigerator and prepared freshly in sodium carboxy methyl cellulose (SCMC) solution just before the experiments.
The rats were divided into six groups containing five rats in each group. Acute inflammation was induced16 by 0.1 ml of 1.0% of λ-carrageenan in normal saline (0.9% w/v NaCl) was injected to the sub plantar region of left hind paw. The extract was administered once to the rats 1 hr before λ-carrageenan injection.
Group I: λ -Carrageenan + Vehicle
Group II: λ -Carrageenan + Aspirin (100 mg/kg. P.o.)
Group III: λ -Carrageenan + Ethyl acetate extract (100 mg/kg. P.o.)
Group IV: λ -Carrageenan + Ethyl acetate extract (200 mg/kg. P.o.)
Group V: λ -Carrageenan + Aqueous extract (100 mg/kg. P.o.)
Group VI: λ -Carrageenan + Aqueous extract (200 mg/kg. P.o.)
The paw volume was measured at 3 hrs after λ -carrageenan injection with the help of plethysmometer. The anti inflammatory activity was evaluated based on the ration of the changes in paw diameter in treated and untreated groups as per the formula given below:
Anti inflammatory activity (%) = [1-(Vt/Vc)] × 100
Vtand Vc are edema volume in drug treated and control groups respectively.
Male Wistar albino rats (250-300 g) were subjected to carrageenan and histamine rat paw edema induced inflammation tests (n=5, in each group). All the animals were housed in groups in polypropylene cages and placed in climate controlled central animal house having temperature 22 ± 2°C, relative humidity 60 ± 5%, and a 12 h light/dark cycle (lights on at 08:00 h and off at 20:00 h). The animals were fed standard pellet diet (Amrut, Pranav Agro Industries Ltd, India) and water ad libitum. All the protocols were approved (approval no-SU/DPS/IAEC/1005) by Institutional Animal Ethics Committee (IAEC) of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India.
Aspirin was dissolved in distilled water. While ethyl acetate and aqueous extract were prepared as suspension in distilled water using 0.5% SCMC as the suspending agent. Animals were assigned to different treatment groups (n=5, in each group). The control group received 0.5% SCMC, (1 ml/kg, p.o.), whereas test groups received ethyl acetate and aqueous extract. All drugs, ethyl acetate and aqueous extract were prepared before 1.00hr of experimentation. All the doses of extracts were administered orally.
The acute toxicity study was performed as per the method described by Litchfield and Wilcoxon (1949) and LD50 was calculated accordingly. Briefly, the Ethyl acetate and aqueous extract in the dose range of 10-1600 mg/kg were administered orally to different groups of mice (n = 10). The animals were examined every 30 min up to a period of 3 h and then, occasionally for additional period of 4 h; finally, overnight mortality was recorded. All tests on rats were performed at three dose levels 100 and 200 mg/kg, p.o. body weight corresponding to 10 and 20% of LD50 value (1000 mg/kg, i.p.), respectively.
All the data were expressed as mean ± SEM from five animals. The data obtained was analyzed using the one-way ANOVA followed by Dunnett Multiple Comparisons Test for determining the level of significance and p < 0.05 was considered statistically significant.
The acute toxicity studies showed that the LD50 of the ethyl acetate and aqueous extracts in mice was 1000 mg/kg by i.p. route. Preliminary phytochemical tests indicated the presence of flavonoids and steroids in the plant.
As compared to the control group, the pretreatrment with ethyl acetate and aqueous extract of M. nagi at (100 and 200 mg/kg, p.o.) showed significant % inhibition against the carrageenan induced rat paw edema after 3 hour as shown in table 1. The M. nagi at 200 mg/kg shows % inhibition similar to those of the standard group. In the same experimental conditions, the carrageenan induce test of the reference drug aspirin (100 mg/kg) was clearly evident (p < 0.01).
As compared to the control group, the ethyl acetate and aqueous extract of M. nagi bark showed (100 and 200 mg/kg, p.o.) showed significant % inhibition (p < 0.01) against the histamine induced rat paw edema after 3 hour as shown in table 2. In the same experimental conditions, the histamine induced test of the reference drug aspirin (100 mg/kg) was clearly evident (p < 0.01).
M. nagi is traditionally used for the treatment of liver diseases, fever, asthma, anemia, chronic dysentery, ulcer and inflammation9,13. Scientific data on these properties of the plant are not available. Therefore, we investigated the effects of different doses of ethyl acetate and aqueous extract using carrageenan and histamine induces rat paw edema. The inflammation consists of three stages, which is increase in vascular permeability, leukocyte migration and proliferation of connective tissue. Swelling caused by carrageenan includes leukocyte migration. It is believable that histamine and serotonin type swelling is the first stage and carrageenan type swelling is the second stage in the inflammatory process. Although the developmental process of acute inflammation the first stage is independent of the second, that is, the first stage is not an indispensable stage to further development of inflammatory reaction. Therapeutic effects of agents on the second stage are however influenced by the first stage11.
Inflammatory events involve micro vascular changes with increased vascular permeability, flow of exudation, including plasmatic protein and amplification of endogenous chemical mediators. Non Steroidal anti inflammatory drugs (NSAIDs) are the common drugs against superficial inflammation. NSAIDs alleviate the hyper allergic symptoms associated with inflammation by inhibiting the COX enzyme and the resultant inhibition of prostaglandins synthesis from arachidonic acid which involved in the pathogenesis of acute inflammation5. This process is mediated by the release of inflammatory paracrine agents such as histamine, neuropeptides, kinins, nitric oxide and PGs, which act as vasodilators. It is now recognized that there are two COX isoforms, COX-I and COX-II3.
Carrageenan injection induced the production of histamine, bradykinin, vasodilatation, PG and cytokine such as TNF-α which causes mucus secretion and mucosal edema, all features of asthma. TNF-α is one of the major inflammatory cytokines with the capacity to prime activation of the neutrophils for their various functions10. These effects were probably due to increased bradykinin levels resulting from the drug′s ability to block kininase II. Bradykinin has been shown to be a mediator of carrageenan inflammation in the rat1. M. nagi plant extracts may reduce the level of histamine, bradykinin, vasodilatation and prostaglandin factors which are responsible in development of inflammation.
Histamine induced paw edema is said to occur in earlier stage in mounting of vascular reaction in the chemically induced inflammation. In this, swelling occurs primarily due to action of histamine. Generally histamine is released following the mast cell degranulation by number of inflammatory mediators including substances P and IL-1. This is likely to evoke the release of neuropeptides as well as release of prostaglandins and monohydroxy eicosatetranoic acid from endothelial cell leading to hyperalgesia and other pro inflammatory effects15. M. nagi plant extracts may act on the inflammatory mediators and inhibit the release of prostaglandins and histamine mediators which causes mucus secretion and mucosal edema.
The present study for the first time provides evidence for the anti inflammatory activity of ethyl acetate and aqueous extract in experimental animals. The presence of flavonoids and steroids in ethyl acetate and aqueous extract could be responsible for these activities. The need of the hour is to identify and isolate the phytoconstituents responsible for the observed central effects in animals and to understand their molecular mechanisms.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. 17
We are grateful to the Head, Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat, India for providing the facilities during the course of this study. Special thanks to Prof. P. Parmar, Botanical Survey of India for identification and authentication of the plant.