Evaluation of Acute and Chronic Anti-Inflammatory Activity of Whole Plant of Calotropis gigantea in Various Experimental Animal Models

 

V.M. Shastry1*, V.R. Patil2 and R.Y. Chaudhari2

1JZMDS’s College of Pharmacy, Mamurabad, Jalgaon-425001, India         2Tapi Valley Education Society’s Hon. LMC College of Pharmacy, Faizpur, Tal- Yawal, Dist-Jalgaon-425503,

*Corresponding Author E-mail: vms30971@rediffmail.com

 

ABSTRACT:

Calotropis gigantea a wild growing plant of family Asclepiadaceae, well known as Sweat akand, is used in traditional medicine to treat inflammation and pain. The present study enumerates investigation of acute as well as chronic anti-inflammatory activity of ethanolic extract of whole plant of Calotropis gigantea (CGEE, 100, 200 and 400 mg/kg, p. o.) in carrageenan- and formalin - induced paw edema and turpentine oil-induced granuloma pouch in rats. Oral administration of CGEE (200 and 400 mg/kg) significantly reduced the paw volume (P <0.001) at 3h in carrageenan model. The treatment of CGEE (200 and 400 mg/kg) significantly reduced the volume of exudates (P <0.001) in turpentine oil-induced granuloma pouch dose dependently. Chronic inflammation induced by formalin injection was significantly (P <0.001) inhibited by CGEE (400 mg/kg) as compared to the control rats. Diclofenac (10 mg/kg) was used as reference drug. Thus, the present study reveals that whole plant of Calotropis gigantea possess significant anti-inflammatory activity and supports the claim in traditional medicine for the treatment of inflammatory conditions. This may be due to the potentiation of its inhibitory effect on synthesis and release of different inflammatory mediators.

 

KEYWORDS: Calotropis gigantea, anti-inflammatory, carrageenan, paw edema, granuloma pouch

 

 


INTRODUCTION:

Herbal medicines are also in great demand in the developed world for primary health care because of their efficacy, safety and lesser side effects. They also offer therapeutics for age-related disorders like memory loss, osteoporosis, immune disorders, etc. for which no modern medicine is available. India despite its rich traditional knowledge, heritage of herbal medicines and large biodiversity has a dismal share of the world market due to export of crude extracts and drugs1.

 

Injurious stimuli cause a protective vascular connective tissue reaction called “inflammation”. Cell injury may occur due to trauma, genetic defects, physical and chemical agents, tissue necrosis, foreign bodies, immune reactions and infections. There are two types of inflammation: acute and chronic. Acute inflammation is characterized by a rapid onset and short duration. It manifests with exudation of fluid and plasma proteins, and emigration of leukocytes, most notably neutrophils.

 

Chronic inflammation is of prolonged duration and manifests histologically by the presence of lymphocytes and macrophages and results in fibrosis and tissue necrosis. When inflammation continues for prolonged periods of time, it can be thought of as the healing process in overdrive, and deleterious changes can occur to localized tissues as well as the entire body2.

 

Every year a lot of plants from traditional medicinal system have been studied for their potential anti-inflammatory and analgesic activity but only few of them only included in health care system after clinical research. Presently used anti-inflammatory drugs are associated with several adverse effects. Therefore efforts shall be taken for the development of newer anti-inflammatory drugs with fewer or very less side effects.

 

The Calotropis gigantea is a perennial shrub belonging to the Asclepiadaceae family found chiefly in wastelands throughout India, in comparatively drier and warmer areas, up to an altitude of 1050 meters3. It is called as “Ruvi” in Marathi and “Madar” in Hindi. Flowers are regular, bisexual, arranged in simple or rarely compound cymose corymbs4. It has been reported traditionally for antifertility, alexipharmic, anthelmintic, purgative and abortifacient activities. The plant has enormous medicinal properties to cures leprosy, leucoderma, ulcers, tumours, piles, diseases of the spleen, liver and abdomen. In folklore medicines C. gigantea roots are reported for their analgesic, anticonvulsant, anxiolytic and sedative properties4. Previous reports suggest the central nervous system stimulant 1 and pregnancy interceptive potentials of root bark5. The juice is anthelmintic, laxative; cures piles and “kapha”. The milk is bitter, oleaginous, purgative; cures leucoderma, tumours, ascites and diseases of the abdomen. The flowers are astringent, bitter and claimed to cure asthma, eczema, leprosy, secondary syphilis, gonorrhea, ascites, helminthiasis, diarrhoea and jaundice in Ayurveda, Siddha and Unani literature6, 7. The flowers of Calotropis gigantea are reported to have hepatoprotective and analgesic activity in mice8,9. Other experiments demonstrated the anti-inflammatory10, 11 and anti-ulcer activity11.

 

Conventionally Calotropis gigantea is in use as indigenous medicine to treat inflammation and pain and to reduce the fever in India. Whether these claims are valid is a subject of great concern and should be probed scientifically. Therefore, the present study was undertaken with the intent to investigate the anti-inflammatory activity of ethanolic extract of whole plant of Calotropis gigantea using acute and chronic pharmacological experimental models.

 

MATERIAL AND METHODS:

Plant materials and extraction:

Plant materials of Calotropis gigantea were collected from wasteland weeds in Jalgaon, M.S., India. The plant was authenticated and identified by Botanical Survey of India, Pune (Voucher No. VIMSPED2). The plant material was shed dried at room temperature for 10 days, coarsely powdered with the help of a hand grinding mill, and the powder was passed through sieve No. 60. The plant material was extracted with 95% ethanol using soxhlet apparatus6. Later the ethanolic extract of Calotropis gigantea (CGEE) was filtered and concentrated (% yield=11.09) using rotary evaporator. CGEE was stored in refrigerator until used.

 

Animals:

Wistar albino rats of either sex, weighing 200-280 g were made available from National Institute of Bioscience, Pune. Rats were housed in polypropylene cages (five per cage) randomly with paddy husk as bedding. The animals were maintained under standard laboratory conditions at temperature 23 ± 2ºC, relative humidity 55 ± 10 % and 12 h light and dark cycle throughout all the experiments. Animals had free access of water and standard laboratory feed ad libitum. All the experimental procedures and protocols used in this study were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of the institute. Ethical guidelines were strictly followed during all the experiments.

 

Acute Toxicity Study:

Acute toxicity studies were carried out using acute toxic class method as per OECD guideline 42312. A starting dose of 2000 mg/kg body weight of extract of Calotropis gigantea was administered orally to three male rats. The animals were observed for mortality and behavioral changes during 48 h.

 

Evaluation of Anti-inflammatory Activity:

Carrageenan-induced pedal edema in rat:

Animals were divided into five groups of six animals in each. In all groups, acute inflammation was produced by sub-plantar injection of 0.1 ml of freshly prepared 1% suspension of carrageenan in normal saline in the right hind paw of the rats and paw volume was measured plethysmometrically at 0 and 3 h after carrageenan injection. Animals were pretreated either with vehicle (5% gum acacia), or CGEE (100, 200 and 400 mg/kg) or Diclofenac (10 mg/kg) orally 13 two hours before injection. Mean increase in paw volume was measured and percentage inhibition was calculated.

 

Turpentine oil-induced granuloma pouch in rat:

Subcutaneous dorsal granuloma pouch was made in ether anaesthetized rats by injecting 2 ml of air, followed by injection of 0.5 ml of turpentine oil into it14,15. All drugs were administered orally one hour prior to turpentine oil injection and continued for seven consecutive days. On 7th day, the pouch was opened under anesthesia, the amount of exudate was taken out with a syringe, and the volume was measured and compared with those of the control and standard group.

 

Formalin-induced edema in rat hind paw:

0.1 ml of 2% formalin was injected into the sub-plantar area of right hind paw of ether anesthetized rat16. All drugs were given orally one hour prior to formalin injection and continued for 7 consecutive days. Degree of inflammation was measured plethysmometrically on days 1 and 7.

 

Statistical analysis:

The results of the study were expressed as mean ± SEM. ANOVA was used to analyze and compare the data, followed by Dunnet’s test for multiple comparisons. P < 0.05 was considered significant in all experiments.

 

RESULTS:

Phytochemical screening:

Preliminary phytochemical evaluation of CGEE showed the presence of alkaloids, flavonoid glycosides, steroids, saponins, tannins, phenolic compounds, triterpenoids, carbohydrates, gums and mucilages.

Acute toxicity study:

No clinical signs of toxicity and mortality found in acute toxicity studies in CGEE treated animals.

 

Carrageenan-induced pedal edema in rat:

CGEE showed anti-inflammatory effect in carrageenan-induced inflammation as mentioned in Table 1. CGEE (200 and 400 mg/kg) dose dependently significantly reduced the paw volume (P <0.001) as compared to the control rats. Diclofenac also showed similar reduction (P <0.001) of inflammation in rats.

 

 

Table 1: Effect of Calotropis gigantea on carrageenan induced rat paw edema

Drug (mg/kg, p. o.)

Paw volume increase after 3 h (ml)

% of inhibition

Control

1.61±0.12

--

CGEE (100)

1.29±0.07

20

CGEE (200)

1.03±0.05**

36

CGEE (400)

0.86±0.08**

47

Diclefenac (10)

0.52±0.04**

68

n = 5 in each group, values are mean±SEM

*P< 0.05, **P< 0.001 compared to control group (ANOVA followed by Dunnett’s test)

 

 

Turpentine oil-induced granuloma pouch:

Table 2 shows the effect of CGEE in turpentine oil-induced granuloma pouch. The treatment of CGEE (200 and 400 mg/kg) significantly reduced the volume of exudates (P <0.001) in turpentine oil-induced granuloma pouch dose dependently, which was comparable with the effect of diclofenac (P <0.001).

 

Table 2: Effect of Calotropis gigantea on turpentine oil- induced granuloma pouch in rat

Treatment (mg/kg, p.o.)

Volume of exudates(ml)

% of  inhibition

Control

2.68±0.08

--

CGEE (100)

2.39±0.17

11

CGEE (200)

2.05±0.11*

23

CGEE (400)

1.68±0.15**

37

Diclofenac(10)

0.89±0.08**

67

n = 5 in each group, values are mean±SEM

*P< 0.05, **P< 0.001 compared to control group (ANOVA followed by Dunnett’s test)

 

 

Formalin-induced edema:

Formalin-induced pedal edema was significantly (P <0.001) inhibited by CGEE (400 mg/kg) as compared to the control rats. Similarly, diclofenac showed inhibitory action on edema formation. Table 3 shows that CGEE was effective in chronic inflammation.

 

Table 3: Effect of Calotropis gigantea on formalin- induced rat hind paw edema

Treatment (mg/kg, p. o.)

Paw volume increase on day 7 (ml)

% of  inhibition

Control

1.49±0.17

--

CGEE(100)

1.18±0.19

21

CGEE (200)

0.94±0.13*

37

CGEE (400)

0.62±0.16**

58

Diclofenac(10)

0.37±0.18**

76

n = 5 in each group, values are mean±SEM

*P< 0.05, **P< 0.005 compared to control group (ANOVA followed by Dunnett’s test)

 

 

DISCUSSION:

In appreciating the inflammatory process, it is important to understand the role of chemical mediators. These are the substances that tend to direct the inflammatory response. These inflammatory mediators come from plasma proteins or cells including mast cells, platelets, neutrophils and monocytes/macrophages. They are triggered by bacterial products or host proteins. Chemical mediators bind to specific receptors on target cells and can increase vascular permeability and neutrophil chemotaxis, stimulate smooth muscle contraction, have direct enzymatic activity, induce pain or mediate oxidative damage. Most mediators are short-lived but cause harmful effects. Examples of chemical mediators include vasoactive amines (histamine, serotonin), arachadonic acids (prostaglandins, leukotrienes) and cytokines (tumor necrosis factor and interleukin–1)17.

 

Most extensively used technique for screening of anti-inflammatory agents is carrageenan-induced inflammation in rats. The development of carrageenan- induced edema is bi-phasic18, in the first phase the release of cytoplasmic enzymes, histamine and serotonin from the mast cells occurs, while in the second phase increased release of prostaglandins in the inflammatory area takes place and kinins provide continuity between the two phases. Since paw edema induced by carrageenan in the second phase is inhibited by CGEE significantly, the present study suggests a possible inhibition of cyclooxygenase synthesis by CGEE, as the carrageenan inflammatory model fundamentally reflects the action of prostaglandins19, 20.

 

Granuloma pouch technique was bespoke15 and turpentine oil is used as irritant. Aseptic inflammation consequential in large volume of hemorrhage exudate was elicited which resembled the sub acute type of inflammation. Turpentine oil-induced granuloma pouch propose a model for exudative type of inflammation. Despite the fact that, the chemical mediators of this type of response are unknown, protein synthesis is obligatory for the formation of granuloma21. CGEE has shown prospective inhibitory action on exudates formation. Kinin is believed to be the main mediator of granuloma, as it not only vasodilate but increase the vascular permeability in the premature stages of inflammation also. Thus, CGEE may own anti-kinin like activity.

 

The most suitable method to monitor antiarthritic and anti-inflammatory agents is likely to be the formalin-induced paw edema assay, as it closely resembles human arthritis. Also, formalin-induced arthritis is a model used for the evaluation of an agent with possible anti-proliferative activity. This experiment is associated with the proliferative phase of inflammation. Result indicates that CGEE may be useful for chronic inflammatory diseases like arthritis as it appears to be effective against formalin-induced edema.

 

Saponin22, terpenoids, flavonoids and steroids contents 23 of many plants have been credited for their anti-inflammatory activities. We deem that the responsibility for anti-inflammatory effect could be mostly due to the presence of saponin, terpenoids, flavonoids and steroids which are the major components. Thus, the current study reveals that whole plant of Calotropis gigantea own significant anti-inflammatory activity and supports the claim in time-honored traditional medicine for the cure of inflammatory conditions.

 

 

REFERENCES:

1.        Kamboj VP. Herbal medicine. Current Science 2000; 78: 35-39.

2.        Jo. Ann R. Gurenlian, the Relationship between Oral Health and Systemic Disease. Special supplemental issue- April 2006. Access: 1-12. Available from: http://www.adha.org/downloads/sup_inflammation.pdf

3.        Argal A and Pathak AK. CNS activity of Calotropis gigantea roots. Journal of Ethnopharmacology, 106:2006, 142–5.

4.        Nadkarni KM. Indian Materia Medica, 3rd revised and enlarged edn, vol 1, Mumbai: Popular Prakashan Private Ltd: 237-246, 1976.

5.        Srivastava SR, et al. Pregnancy interceptive activity of the roots of Calotropis gigantea Linn. in rats. Contracep, 2007, 75: 318– 22.

6.        Chitme HR, et al. Studies on anti-diarrhoeal activity of Calotropis gigantea R.Br. in experimental animals. Journal of Pharmacy and Pharmaceutical Sciences, 2004, 7(1): 70-75.

7.        Kirtikar KR and Basu BD. Indian Medicinal Plants, 2nd ed, vol 3, Allahabad, India: Lalit Mohan Basu: 1607-11, 1998.

8.        Pathak AK and Argal A, Analgesic activity of Calotropis gigantea flower. Fitoterapia, 2007, 78: 40–2.

9.        Tenpe CR, et al. Screening of methanolic extract of Calotropis gigantea leaves for hepatoprotective activity. Indian Drugs, 2007, 44(11): 874-5.

10.     G.S. Pardesi, et al. Evaluation of anti-inflammatory activity of Calotropis gigantea. Pharmacologyonline, 2008, 1: 111-6.     

11.     Kshirsagar A, et al. Anti-inflammatory and anti-ulcer effect of Calotropis gigantea R. Br. Flower in rodent. Journal of Natural Remedies, 2008, 8(2): 183-90.

12.     OECD: acute oral toxicity- Acute toxic class method guidelines 423. Paris, 1996.

13.     Tonussi CR and Ferreira SH. Mechanism of diclofenac analgesia: direct blockade of inflammatory sensitization. European Journal of Pharmacology; 1994, 251:173-179.

14.     Selye H. On the mechanism through which hydrocortisone affects the resistance of tissue to injury. An experimental study with granuloma pouch technique. Journal of American Medical Association; 1953, 152:1207-1213.

15.     Robert A and Nezamis JE. The granuloma pouch as a routine assay for anti-phlogistic compounds. Acta Endocrinologica; 1957, 25: 105-107.

16.     Chau TT. Analgesic testing in animal models. In: Pharmacological methods in the control of inflammation. Alan R Liss Inc., 1989, 195-212.

17.     Mariotti A. A primer on inflammation. Compend. Cont. Educ. Dent. 2004; 25 (7) (Suppl 1):7-15.

18.     Larsen GL and Henson PM. Mediators of inflammation. Ann. Rev. Immunol; 1983, 1:335-5.

19.     Brooks PM and Day RO. Nonsteroidal anti-inflammatory drugs: differences and similarities. N Engl J Med; 1991, 324:1716-1725.

20.     Vane J and Booting R. Inflammation and the mechanism of action of anti-inflammatory drugs. FASEB Journal; 1987, 1:89-96.

21.     Selye H. Use of "Granuloma Pouch" technique in the study of antiphlogistic corticoids. Proc Soc Exp Biol Med; 1953, 82:328-333.

22.     Oweyele VB, et al. Analgesic and anti-inflammatory properties of Nelsonia canescens leaf extract. Journal of Ethnopharmacol; 2005, 99:153-156.

23.     Adedapo AA et al, Anti-inflammatory and analgesic activities of the aqueous extract of Cussonia paniculata stem bark. Rec Nat Prod; 2008, 2(2):46-53.

 

 

 

 

 

 

Received on 19.03.2011          Modified on 23.03.2011

Accepted on 28.03.2011         © RJPT All right reserved

Research J. Pharm. and Tech. 4(5): May 2011; Page 815-818