|Year : 2012 | Volume
| Issue : 6 | Page : 788-791
Evaluation of anti-inflammatory activity of Typha angustifolia pollen grains extracts in experimental animals
Saroj S Varpe1, Archana R Juvekar1, Mukta P Bidikar2, Parikshit R Juvekar3
1 Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga, Mumbai, India
2 Department of Physiology, Topiwala National Medical College & BYL Nair Charitable Hospital, Mumbai Central, Mumbai, India
3 Department of Physiology, K. J. Somaiya Medical College, Sion, Mumbai, India
|Date of Submission||09-Feb-2012|
|Date of Decision||27-Aug-2012|
|Date of Acceptance||31-Aug-2012|
|Date of Web Publication||8-Nov-2012|
Archana R Juvekar
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga, Mumbai
Source of Support: UGC, Conflict of Interest: None
Objective: This study was designed to evaluate the anti-inflammatory activity of aqueous and 70% methanolic extracts of pollen grains of Typha angustifolia.
Materials and Methods: Female Sprague Dawley rats were used for the study. The acute anti-inflammatory activity of pollen grains of T. angustifolia was studied using the carrageenan as phlogistic agent, whereas its chronic anti-inflammatory effect was investigated by the percentage inhibition of cotton pellet-induced granuloma.
Results: Both aqueous and 70% methanolic extracts of pollen grains of T. angustifolia showed significant dose-dependent inhibition of carrageenan-induced paw edema as compared to the control (P<0.001). It was observed that both the extracts at dose of 125 mg/kg inhibited the granuloma formation by 44.30% which is higher than at dose of 500, 250 mg/kg, thus causing a significant (P<0.001) non-dose-related inhibition of granuloma formation.
Conclusion: The results of this study indicate that extracts of pollen grains of T. angustifolia are effective in the treatment of both acute and chronic inflammatory conditions and thus support its traditional utilization.
Keywords: Anti-inflammatory, carrageenan, cotton pellet-induced granuloma, Typha angustifolia
|How to cite this article:|
Varpe SS, Juvekar AR, Bidikar MP, Juvekar PR. Evaluation of anti-inflammatory activity of Typha angustifolia pollen grains extracts in experimental animals. Indian J Pharmacol 2012;44:788-91
|How to cite this URL:|
Varpe SS, Juvekar AR, Bidikar MP, Juvekar PR. Evaluation of anti-inflammatory activity of Typha angustifolia pollen grains extracts in experimental animals. Indian J Pharmacol [serial online] 2012 [cited 2021 Mar 6];44:788-91. Available from: https://www.ijp-online.com/text.asp?2012/44/6/788/103303
| ╗ Introduction|| |
Inflammation is a body defense mechanism which eliminates or limits the spread of the injurious agent. It serves to destroy, dilute, or wall off the offender and sets into motion a series of events that try to heal and reconstitute the damaged tissue. When tissue injury occurs, whether caused by bacteria, trauma, chemicals heat, or any other phenomenon, multiple substances are released by the injured tissues which cause dramatic secondary changes in the surrounding uninjured tissues.  Although effective, all the steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs), cause undesired and serious side effects. Hence, the development of a safer and highly efficacious alternative is still needed.
The traditional uses of medicinal plants are being substantiated by research and identification of the active chemical constituents. Typha angustifolia of the family Typhaceae is commonly known as Elephant grass or Cattail. It is used in treatment of leprosy.  The wooly soft inflorescence is hemostatic and used for dressing ulcers and wound healing. Folkloric uses of pollen grains of T. angustifolia are kidney stones, dysmenorrhea, abnormal uterine bleeding, abscesses, and also tapeworm infestation, diarrhea, and dysentery.  Modern studies have found that pollen grains of Typha mainly contain sterols, terpenoids, flavanoid glycosides,  cerebrosides, and long chain hydrocarbons that possess various pharmacological activities like immunosuppression,  antiplatelet aggregation,  antimicrobial activity,  cholesterol lowering, and antiatherogenic effect.  This study was conducted to investigate the anti-inflammatory activity of aqueous and 70% methanolic extracts of T. angustifolia using acute and chronic experimental models of inflammation.
| ╗ Materials and Methods|| |
Procurement of the Plant
Pollen grains of T. angustifolia were collected from Goregaon and authenticated by Dr. Ganesh Iyer, Department of Botany, Ruia College, Matunga, Mumbai. The pollen grains were then dried using tray dryer for further studies.
λ-Carrageenan was procured from Sigma Aldrich (St. Louis, MO, USA). Standard drugs such as indomethacin (INDOCAP 25 mg, Jagson Pal Pharmaceutical Ltd., New Delhi, India) and diclofenac (REACTIN-50, Cipla Ltd., Haridwar, Uttarakhand, India) were purchased from Matunga, Mumbai.
Preparation and Screening of Plant Extract
Dried pollen grains of T. angustifolia were refluxed with distilled water and 70% methanol for 5 h at 70-80°C twice, to produce aqueous and methanolic extracts, respectively. Both the extracts were concentrated, which were then subjected to phytochemical evaluation using qualitative chemical tests for detecting the presence of the phytoconstituents like alkaloids, glycosides, tannins, phenolic compounds, phytosterols, carbohydrates, proteins, and amino acids.
Identification of Active Compound Using Thin Liquid Chromatography
The 70% methanolic extract of pollen grains of T. angustifolia was fractionated using chloroform. This fraction was examined separately on silica gel 60 F 254 precoated plates using toluene :0 methanol (8:2) as a chromatographic solvent. The developed chromatogram was heated for 10 min at 105°C and spots were revealed by Lieberman Burchard reagent. The active component of chloroform fraction was β-sitosterol (Rf : 0.605) which was confirmed with standard β-sitosterol.
Determination of Total Phenolics and Flavanoids
Total phenols were estimated as gallic acid equivalents (GAE), expressed as mg gallic acid /g extract . To 6 ml of H 2 O, 100 μl of the sample was transferred in a 10 ml volumetric flask, and 500 μl undiluted Folin-Ciocalteu reagent was subsequently added. After 1 min, 1.5 ml 20% (w/v) Na 2 CO 3 was added and the volume was made up to 10 ml with H 2 O. After 2 h incubation at 25°C, the absorbance was measured at 760 nm and compared to a gallic acid calibration curve. The data presented are the average of triplicate analyses. 
Total flavanoids were estimated as rutin equivalents, expressed as mg rutin /g extract . One ml of the plant extract in methanol (10 g/L) was mixed with 1 ml aluminum trichloride in ethanol (20 g/L), which was then diluted with ethanol to 25 ml. Absorbance at 415 nm was read after 40 min at 20°C. Blank samples were prepared from 1 ml plant extract and one drop acetic acid, and diluted to 25 ml. The rutin calibration curve was prepared in ethanolic solutions with same procedure. All determinations were carried out in quadruplicate and the mean values were used.
Female Sprague Dawley rats (200-250 g) were procured from Glenmark Pharmaceuticals, Mhape, Navi Mumbai. The animals were placed in polypropylene cages in a controlled room with temperature 22±1°C and relative humidity of 60-70% (87/1999/CPCSEA). They were given a standard pellet diet (Amrut Brand, Sangli, India) and water ad libitum. The animals were acclimatized to laboratory conditions for 7 days before commencement of experiment. Ethical clearance was obtained from Institutional Animal Ethics Committee.
Acute Toxicity Study
Acute oral toxicity studies for the extracts of pollen grains of T. angustifolia were carried out as per the Organization for Economic Co-operation and Development (OECD) guideline no. 423. The acute toxic class method set out in this guideline is a stepwise procedure with the use of three animals of the same gender per step. Depending on the mortality and/or the moribund status of the animals, on average 2-4 steps may be necessary to allow judgment on the acute toxicity of the test substance.
Carrageenan-induced Rat Paw Edema
The female Sprague Dawley rats were divided into eight groups of six animals each. The first group (control) received 0.5% sodium carboxymethyl cellulose (CMC) and the second group (positive control) received 10 mg/kg p.o. indomethacin as a standard. The remaining groups received aqueous (groups III, IV, and V) and 70% methanolic extracts (groups VI, VII, and VIII) at a dose of 500, 250, and 125 mg/kg orally. After 1 h, the rats were challenged with a subcutaneous injection of 0.1 ml of 1% w/v solution of λ-carrageenan into the plantar side of the left hind paw. The left paw was marked with ink at the level of lateral malleolus and immersed in mercury up to the mark. The paw volume was measured with a plethysmograph before, immediately after (0 h), and then every hour till 5 h after injection of carrageenan. 
Inflammation was assessed as the difference between the baseline volume of the treated paw (V0 ) and the volume at various times (Vt ) after injection of the carrageenan and the percent inhibition of edema was calculated using the relation:
Inhibition of edema (%) =100×[1−(a−x/b−y)],
where a is the mean paw volume of treated rats at various time after carrageenan injection; x, the mean paw volume of treated rats before carrageenan injection; b, the mean paw volume of control rats at various time after carrageenan injection; and y, the mean paw volume of control rats before carrageenan injection.
Cotton Pellet-Induced Granuloma
The effect of the extract on chronic inflammation was evaluated using cotton pellet-induced granuloma in rats. Female Sprague Dawley rats were divided into 10 groups of six animals each. The first group (control) received 0.5% sodium CMC and the second group (positive control) received 20 mg/kg p.o. diclofenac as a standard. The remaining groups received aqueous (groups III, IV, V, and VI) and 70% methanolic extracts (groups VII, VIII, IX, and X) at a dose of 500, 250, 125, and 75 mg/kg orally. The extracts were administered once daily for 7 days. Thirty minutes after giving the first dose of the respective treatments (control, standard, or extract), two autoclaved cotton pellets (30±1.0 mg) were aseptically implanted under the previously depilated back of anaesthetized rats. On day 8, the animals were sacrificed. The pellets were dissected out, freed of tissue attachments, and dried in the oven overnight at 60°C. The dry pellets were weighed and the mean weight of the granuloma tissue formed around each pellet was determined. The level of inhibition of granuloma tissue development was calculated using the relation:
Inhibition=100×(Tc−Tt /Tc ),
where Tc is the weight of granuloma tissue of control group and Tt is the weight of granuloma tissue of treated group. 
The observations were expressed as mean±SD and the difference in response to test drugs and control was determined by one-way analysis of variance (ANOVA) followed by Dunnett's t-test. P <0.05 was considered as statistically significant.
| ╗ Results|| |
Phytochemical Screening of the Extracts
As shown in [Table 1], the preliminary phytochemical evaluation of both aqueous and methanolic extracts using qualitative chemical tests showed presence of carbohydrates, flavanoids, phenolic compounds and only the methanolic extract tested positive for sterols and terpenoids. The content of total phenols and total flavanoids were relatively more in 70% methanolic extract than aqueous extract. Chloroform fraction of 70% methanolic extract of T. angustifolia as revealed by TLC showed presence of sterols as experimental Rf value of chloroform fraction (0.605), matched with standard β-sitosterol.
|Table 1: Phytochemical analysis of aqueous and 70% methanolic extracts of pollen grains of Typha angustifolia|
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Acute Oral Toxicity Study
In the acute oral toxicity study, no deaths were observed at the dose tested. The oral LD 50 was determined to be higher than highest dose tested, i.e., 2000 mg/kg.
Carrageenan-induced Rat Paw Edema
When compared with the control group, the aqueous extract of T. angustifolia at concentrations of 500, 250, and 125 mg/kg inhibited the edema formation by 78% (P <0.001), 69% (P <0.001), and 70% (P <0.001), respectively, in a dose-dependent manner at the 3 rd h and this inhibition decreased thereafter. The 70% methanolic extract at concentrations of 500, 250, and 125 mg/kg inhibited the edema formation by 62% (P <0.001), 58% (P <0.001), and 53% (P <0.01), respectively, in a dose-dependent manner at the 3 rd h and this inhibition slightly decreased thereafter. The standard drug indomethacin (10 mg/kg) significantly inhibited the edema formation by 94% and 100% at 3 rd and 5 th h, respectively (P <0.01), as compared with control group [Table 2].
|Table 2: Effect of aqueous and 70% methanolic extracts of Typha angustifolia pollen grains on carrageenan-induced paw edema in rats|
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Cotton Pellet-Induced Granuloma
When compared with the control group, the aqueous extract inhibited the granuloma formation by 35.5% (P <0.001), 36% (P <0.001), 44.3% (P <0.001), and 38.6% (P <0.001) at concentrations of 500, 250, 125, and 75 mg/kg, respectively. The 70% methanolic extract inhibited the granuloma formation by 23.1% (P <0.001), 36% (P <0.01), 44.3% (P<0.001), and 36.5% (P<0.001) at concentrations of 500, 250, 125, and 75 mg/kg, respectively. The standard drug diclofenac (20 mg/kg) significantly inhibited the granuloma formation by 68.5% when compared with the control group [Table 3].
|Table 3: Effect of aqueous and 70% methanolic extracts of Typha angustifolia pollen grains on cotton pellet-induced granuloma in rats|
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| ╗ Discussion|| |
Preliminary phytochemical screening of aqueous and 70% methanolic extracts of pollen grains of T. angustifolia revealed the presence of carbohydrates, proteins, phenolic compounds, flavanoids, and sterols. Chromatographic investigation of the methanolic extract proved the presence of β-sitosterol, which is known to possess anti-inflammatory activity and may account for the efficacy of the extract in the animal models. Extracts of T. angustifolia were found to be safe up to the dose of 2000 mg/kg since no mortality and abnormal behavioral patterns were observed.
Carrageenan-induced inflammation is useful in detecting orally active anti-inflammatory agents. Edema formation due to carrageenan is a biphasic event.  The initial phase is attributed to the release of histamine and serotonin. The edema produced at the peak (3 h) is thought to be due to the release of kinin-like substances, especially bradykinin. The second phase of edema is due to the release of prostaglandins, protease, and lysosome. The second phase is sensitive to most clinically effective anti-inflammatory drugs. The reduction in the paw edema was significant during the first phase of inflammation, indicating the inhibition of histamine release as well as during the second phase, which may due to the inhibition of cyclooxygenase enzymes involved in the formation of prostaglandins. Pretreatment with both aqueous and 70% methanolic extracts showed significant dose-dependent decrease in rat paw edema when compared to control group.
The cotton pellet granuloma method has been widely employed to assess the transudative, exudative, and proliferative components of chronic inflammation. The fluid absorbed by the pellet greatly influences the wet weight of the granuloma and the dry weight correlates well with the amount of granulomatous tissue formed.  Monocyte infiltrations and fibroblast proliferation, rather than neutrophil infiltrations and exudation, take place in chronic inflammation. In this study, the aqueous and 70% methanolic extracts of T. angustifolia decreased dry weight of the cotton pellets compared to control groups. The aqueous and 70% methanolic extracts at dose of 125 mg/kg inhibited the granuloma formation by 44.30% which is higher than at dose of 500, 250 mg/kg which demonstrates a significant (P<0.05) non-dose-related inhibition of granuloma formation. This may be due to the ability of T. angustifolia in reducing the number of fibroblasts and synthesis of collagen and mucopolysaccharide, which are natural proliferative agents of granulation tissue formation.
This study revealed the in vivo anti-inflammatory activity of aqueous and 70% methanolic extracts of T. angustifolia and the presence of flavanoids and sterols may be responsible for the activity. Further investigations are required to find active components of the extracts and to confirm the mechanism of action.
| ╗ Acknowledgments|| |
Authors are thankful to University Grant Commission, Govt. of India, New Delhi for financial support.
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[Table 1], [Table 2], [Table 3]
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