|Year : 2021 | Volume
| Issue : 1 | Page : 103-108
Anti-inflammatory potential of aqueous extract of Elsoltzia stachyodes on experimental models of inflammation in rats
Priyotosh Banerjee1, Tarun Kumar1, Sudhir Chandra Sarangi2, Usham Dharmaraja Meetei1, Akham Subhalakshmi Devi1, Ritesh Kumar1
1 Department of Pharmacology, Regional Institute of Medical Sciences, Imphal, Manipur, India
2 Department of Pharmacology, All India Institute of Medical Sciences, New Delhi, India
|Date of Submission||15-Jan-2020|
|Date of Decision||18-Feb-2020|
|Date of Acceptance||30-Mar-2020|
|Date of Web Publication||27-Jan-2021|
Department of Pharmacology, Regional Institute of Medical Sciences, Lamphelpat, Imphal - 795 004, Manipur
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: Elsholtzia stachyodes is being used as a folk medicine to suppress the pain in various inflammation-related disorders in the North-eastern part of India. However, there is a lacking of evidence-based data on it. Hence, this study was conducted to evaluate the anti-inflammatory properties of an aqueous extract of Elsoltzia stachyodes (AEES) on experimental models of inflammation in rats. Materials and Methods: The qualitative phytochemical analysis tests were performed to check the presence of alkaloids, glycoside, flavonoids, saponin, tannin, and triterpenoids. Wistar (150–175 g) rats were used to evaluate anti-inflammatory property by carrageenan induced paw edema and cotton pellet-induced granulation tissue formation models. Indomethacin (10 mg/kg) and AEES were administered at doses of 50, 100, and 200 mg/kg in both the models. In case of carrageenan-induced paw edema model, the change in paw volume was estimated using plethysmometer. However, wounds weights of cotton pellet were measured in cotton pellet-induced granulation tissue formation model. Results were analyzed by one-way ANOVA. Results: The phytochemical analysis of AEES showed the presence of flavonoids, saponin, tannin, and triterpenoids. AEES showed a significant decrease in paw edema and weight of the cotton pellets in a dose-dependent manner in both the inflammatory models. Conclusion: The findings of the present study suggest that AEES exhibited significant anti-inflammatory activity and may have the potential of usefulness as an adjuvant therapy in inflammatory-related disorders.
Keywords: Elsholtzia stachyodes, granulation tissue, inflammation, paw edema, rat
|How to cite this article:|
Banerjee P, Kumar T, Sarangi SC, Meetei UD, Devi AS, Kumar R. Anti-inflammatory potential of aqueous extract of Elsoltzia stachyodes on experimental models of inflammation in rats. J Nat Sc Biol Med 2021;12:103-8
|How to cite this URL:|
Banerjee P, Kumar T, Sarangi SC, Meetei UD, Devi AS, Kumar R. Anti-inflammatory potential of aqueous extract of Elsoltzia stachyodes on experimental models of inflammation in rats. J Nat Sc Biol Med [serial online] 2021 [cited 2021 Jul 29];12:103-8. Available from: http://www.jnsbm.org/text.asp?2021/12/1/103/307855
| Introduction|| |
Inflammation is a local response of mammalian tissue in response to any injury caused by any agent, characterized by pain, swelling, redness, and production of heat. It is a protective response of the body against various noxious stimulations but sometimes become unbearable. Lots of medications have been invented to suppress the inflammation, but most of them have prominent side effects such as gastritis, renal problem, and hepatoxicity. On the other hand, the cost of medicine is the major challenge for developing countries, such as South Asian and African countries. This emerges the importance of developing new and alternative drugs which have less chance of developing disastrous adverse effects on affordable cost.
Since long past, Indian people are using herbal products for treating a variety of diseases. Beginning from sixth century B. C. Charaka Samhita contains the first-ever documentation of Ayurveda, i.e., recordings of treatment by natural products. The uses of herbal products have been increased dramatically in the last decade as do have lesser side effects and being cheaper it is easily accessible to common man, particularly in developing countries like India. Herbal medicines for the treatment of inflammation are very popular in developed and developing countries., Anti-inflammatory activity of plant extracts belongs to the Indian traditional medication system has been documented such as Terminalia chebula, Terminalia bellirica, Psidium guajava, Pistacia atlantica.
Elsoltzia stachyodes commonly known as Vietnamese balm is found abundantly in the Himalayan region, and also in North-eastern India, Myanmar, at an altitude of 800–1800 m from sea level. It has been used by the North-eastern people in the treatment of viral and bacterial infection, fever, pain, and stomach disorder. The other plants of Elsholtzia have a wide range of utility in many disease conditions in local remedies as well as food products. The antioxidant and anti-inflammatory properties of Elsholtzia species have been reported by various studies. However, it was being well established that natural product with antioxidant property play an important role in the suppression of inflammation with lesser side effects. Hence, the present was planned to evaluate the anti-inflammatory potential of aqueous extract of E. stachyodes (AEES) in the experimental models in rats.
| Materials and Methods|| |
The Wistar rats weighing 150–175 g of either sex is taken from the Central Animal Facility of Regional Institute of Medical Sciences (RIMS), Imphal, India. The rats were housed in polyacrylic cages and kept not more than 4 animals per cage. They were maintained under standard laboratory conditions with natural dark and light cycle (12:12 h) and standard dry rat pellet diet, and tap water was provided ad libitum. The study protocol was approved by the Institutional Animal Ethics Committee of RIMS, Imphal (Reg no. 1596/GO/a/12/CPCSEA).
The whole plant was collected from the Imphal valley in between September and October month. Authentication of the plant was done by Prof. P. K. Singh, Department of Life Science, Manipur University (Acc. No. MUMP: 0113).
Preparation of the extract
The plants were air-dried under shade and dried plants were powdered by a mixer grinder. The extraction was done using distilled water at 100°C by soxhlet apparatus. The yield of the extract was 14% and stored in an airtight container at 4°C for further use.
The solubility test of AEES, standard drug (Indomethacin), and carrageenan was checked in distilled water and normal saline.
The presence of flavonoids, steroids, alkaloid, saponin triterpenoid, tannin and glycoside was analyzed by the following methods:
Few drops of 5% ferric chloride were added in 2–3 ml of the extract solution of E. stachyodes. The appearance of blue color indicated of hydrolysable tannins and green color indicated condensed tannins.
Diluted HCL was added in evaporate aqueous extract residue of E. stachyodes. The mixture was shaken and filtered it. After that, 2–3 drops of Wagner's reagent was added into the 2–3 ml of the filtrate. The appearance of reddish brown precipitate indicates the presence of alkaloids.
Few drops of lead acetate were added in 2 ml of extract solution. Formation of yellow-colored precipitate indicated the presence of flavonoids.
Steroids and triterpenoids
The presence of steroids and triterpenoids was analyzed by Salkowsky reaction. In which, 2 ml of concentrated sulfuric acid was slowly added by the side of the test tube containing known volume of extract solution. The presence of steroids was shown if the red color appeared in the lower layer. Similarly, the occurrence of yellow color showed the presence of triterpenoids.
The presence of cardiac glycoside was analyzed by Keller–Killiani test. In which, 0.4 ml glacial acetic acid and 1 drop of 5% ferric chloride solution were added in 2 ml of the extract solution. After that, 0.5 ml of concentrated sulfuric was slowly added in the mixture of extract solution, glacial acetic acid, and ferric chloride solution. The appearance reddish-brown color at the junction of the two layers indicated the presence of cardiac glycoside.
The formation of thick foam after vigorously shaking of the extract solution indicated the presence saponin.
Chemicals and reagents
Indomethacin, carrageenan, and phenobarbitone were procured from standard company HiMedia.
Carrageenan-induced paw edema
The standard procedure for carrageenan induced paw edema model was performed as per the method explained by Medhi and Prakash, 2010. Carrageenan was dissolved in distilled water and administered (0.1 ml of 1%) subcutaneously in the hind paw. The change in paw volume was measured hourly using plethysmometer.
Cotton pellet-induced granuloma pouch
The method was carried out as explained in Mazumder et al., in 2003. Under general anesthesia, a sterile cotton pellet of 5 mg was kept in situ under the skin at the nape of the neck. Increment in its weight was measured after 10 days.
The animals were divided into six groups with 6 animals each, Group 1: Vehicle control 1 ml/100 g of animal weight, Group 2: Carrageenan induced inflammation/cotton pellet-induced granuloma pouch, Group 3: Indomethacin (10 mg/kg), and Groups 4, 5, and 6 were treated with AEES (50, 100, and 200 mg/kg), respectively [Table 1] and [Table 2].
|Table 2: Treatment groups for cotton pellet induced granuloma pouch method|
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Carrageenan induced paw edema method
Animals were weighed and grouped. Test and standard groups were given the drugs 1 h before administration of carrageenan. Carrageenan and vehicle control groups were given only distilled water. The paw volume was measured using a mercury plethysmometer and pretreatment volumes were recorded. After 1 h of drug administration, 0.1 ml of 1% carrageenan solution was injected subcutaneously at the hind paw of each rat except the vehicle control group and paw volumes were recorded 1 h after carrageenan administration. The percentage inhibition was calculated as the following formula:
Cotton pellet induced granulation tissue formation
Nape of the neck of each rat was shaved and cleaned with spirit. A small incision was given under phenobarbital anesthesia and pre weighted sterile cotton pellet of 5 mg each were put subcutaneously. The area is repaired and antiseptic ointment (Betadine) was applied to all groups. Drug treatment was started 1 h after the procedure. Indomethacin group was treated with 10 mg/kg of indomethacin; AEES 50, 100, and AEES 200 groups were treated accordingly. After treatment for 10 days, the wounds were opened and weights of each cotton pellet were measured.
Histological examination of the granulation tissue was done by collecting the granulation tissue obtained from the cotton pellets. Granulation tissues were preserved in 10% formalin. Tissues were stained with hematoxylin and eosin and observed under ×20 magnifications.
Data were analyzed using SPSS version 21.0 (IBM, Armonk, New York) and was expressed as mean ± standard error of mean and analyzed using one-way ANOVA with post hoc tests.
| Results|| |
The AEES, standard drug (indomethacin), and carrageenan were freely soluble in distilled water.
Result for phytochemical analysis
The phytochemical analysis of the AEES showed the presence of flavonoids, saponin, tannin, triterpenoid [Table 3].
|Table 3: Qualitative phytochemical analysis of an aqueous extract of Elsholtzia stachyodes|
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Results for carrageenan-induced paw edema model
There was significantly increased (P < 0.001) volume of rat hind paw after the sub plantar injection of carrageenan in carrageenan group as compared to the vehicle control group. Indomethacin treatment significantly (P < 0.001) ameliorated paw volume as compared to carrageenan group. Similarly, the AEES treatment also decreased the paw volume in dose-dependent manner. At 1 h the max inhibition has been observed in the indomethacin group (38.5% ± 0.81%). In AEES 50, 100, and 200 groups, the inhibition was observed at 4.91% ± 1.26%, 20.94% ± 1.32%, and 25.00% ± 1.07%, respectively [Table 4].
|Table 4: Effect of aqueous extract of Elsholtzia stachyodes treatment on carrageenan induced paw edema in rats|
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Results for granuloma pouch method
The weight of the granulation tissue accumulated in the cotton pellet after 10 days showed the significant decreased weight of granulation tissue in indomethacin (P < 0.001) and AEES (100 and 200 mg/kg) groups (P < 0.001) as compared to the disease control group. The weight of the granuloma pouch found 16.0 ± 0.32 mg in the indomethacin group which was significantly lower than disease control group. The significant reduction in the weight of granuloma pouch was also found in AEES 100 and AEES 200 groups 19.4 ± 0.25 and 17.6 ± 0.25 mg, respectively. However, AEES 50 group did not show significant difference in the weight of the granuloma pouch as compared to the disease control group [Table 5].
|Table 5: Effect of aqueous extract of Elsholtzia stachyodes treatment on the weight of granulation tissue on the granuloma pouch model in rats|
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Result of the histological examination of granulation tissue
In the vehicle control group, inflammatory exudates were accumulated with lots of polymorphs and newly formed blood vessels. In the indomethacin group, there was accumulation of scanty neutrophils, some neovascularization and collagen fibers with few inflammatory cells. However, in the AEES 50 group, lots of polymorphs and neovascularization were seen as the similar to vehicle control group. However, AEES 100 and 200 treated group, only a small number of fibroblasts, polymorphs with few lymphocytes were observed [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5].
|Figure 1: Histological picture of granulation tissue in disease vehicle control group|
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|Figure 2: Histological picture of granulation tissue in Indomethacin group|
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|Figure 3: Histological picture of granulation tissue in aqueous extract of Elsholtzia stachyodes 50 group|
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|Figure 4: Histological picture of granulation tissue in aqueous extract of Elsholtzia stachyodes 100 group|
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|Figure 5: Histological picture of granulation tissue in aqueous extract of Elsholtzia stachyodes 200 group|
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| Discussion|| |
Inflammation is one of the common presentations of various diseases such as tissue injury, arthritis, and gout. Nonsteroidal anti-inflammatory drugs are widely used for its treatment. However, the side effects of these drugs are quite common and scale of various adverse effect changes according to its use. The side effects may appear in the form of gastrointestinal reactions such as gastritis and bleeding, to renal damage. Serious side effects are more common with nonselective COX inhibitors, which alarms for the development of new remedies. The use of medicinal plants and their metabolites are gradually increased for treating inflammation as complementary medicine. The anti-inflammatory potential of various medicinal plants such as Curcuma longa, Zingiber officinale, Rosmarinus officinalis, Mangifera indica have been reported.,,, The anti-inflammatory potential of Elsholtzia plants was well documented. However, there were lacking of evidence-based information on it. Hence, in this study, the anti-inflammatory activity of E. stachyodes has been evaluated on the experimental models inflammation in rats. The carrageenan induced paw edema and granuloma pouch methods are one of well-accepted experimental models to analyze the anti-inflammatory activity of herbal extracts. So, these two were selected in the present study to evaluate the anti-inflammatory activity of AEES. The significant (P < 0.001) augmentation was found in paw edema and weight of cotton pellet in carrageenan induced paw edema model and granuloma pouch model as compared to vehicle control group respectively. The changes in paw edema and cotton pellet weight proved that inflammation was induced in the animals of models. Indomethacin was used as a standard drug in this study. Previously, various studies also used it as a standard drug for the screening of inflammatory activity.,,
Carrageenan and granuloma pouch can induce inflammation by stimulation of arachidonic acid secretion and also provokes the release of pro-inflammatory cytokines such as tumor necrosis factor (TNF) α and interleukin (IL)-1 β. Flavonoids and triterpenoids may suppress the release of arachidonic acid, TNFα and IL-1 β by inhibiting COXs and 5-LOX enzymes., The phytochemical analysis of AEES in the present study found the presence of flavonoids and triterpenoids in the extract. Hence, the anti-inflammatory activity of AEES may be due to the presence of flavonoids and triterpenoids.
The changes in weight of the cotton pellet in the granuloma pouch model may be due to infiltration of macrophages, neutrophils, and proliferation of fibroblast. Significant decreases were observed in AEES-treated groups may be due to suppression of infiltration of these cells. However, the histological examination of the tissue has also demonstrated supportive evidence in this aspect. In which, the control group showed maximum accumulation of inflammatory exudates with lots of polymorphs and newly formed blood vessels. While indomethacin and AEES-treated groups had minimized the inflammatory cells and exudates.
| Conclusion|| |
The findings of the present study demonstrate the anti-inflammatory potential of AEES in the experimental models of inflammation. There is a need for phytochemicals based analysis to find out the compounds which are responsible for its efficacy.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]