Evaluation of Anti-Inflammatory, Antipyretic and Antifungal Activity of Solanum laeve Dunal

 

P Muthumani*1, R Meera1, Sweetlin1, P Devi1, B Kameswari1 and B Eswara Priya2

 

1K.M. College of Pharmacy, Uthangudi, Madurai – 625 107.

2Department of Biotechnology, St. Michael College of Engineering, Sivagangai, TamilNadu, India,

 

 

ABSTRACT

Solanum leave Dunal var biegninatum is small shrub is used by the tribals as a medicinal plant. The objective of the present investigation was to study the anti inflammatory, antipyretic and anti fungal activity potential in experimental animal models. The isolated compounds obtained from the chromatography after the extraction completed were used for (SA-I, SA-II, SA-III) these studies. Anti inflammatory potential of isolated compounds (SA-I, SA-II, SA-III) was evaluated by Carrageenan induced paw edema method in rats. Also antipyretic activity was tested on animal models. The studies were conducted on wistar rats of either sex (160-180g). The change in edema volume of the rat hind paw was measured using plethysmometer. The SA-III of isolated compound of Solanum leave dunal inhibited the formation of edema to significant levels in rats treated with carrageenan. At a dose of 100mg/kg orally, the SA-III produced 40.90% inhibition in case of the carrageenan induced edema (p<0.01).

 

The isolated compounds of Solanum leave Dunal (SA-I, SA-II,SA-III) were also evaluated for antipyretic activity on animals as per Vogel’s method. Solanum leave Dunal elicited a dose dependent inhibition of rectal temperature compared with control group. The isolated compound SA-II having maximum antipyretic activity which was compared with standard inhibition at 300mg/kg p.o.

 

The fungal activity of the isolated compounds (SA-I,SA-II,SA-III) were prepared at various concentration of 1%,2% and 3% solution respectively. In this the compound SA-II(C) having significant antifungal activity compared with other compound against Candida albicans.

 

The results indicated that the isolated compounds (SA-I,SA-II,SA-III) were active against all the experimentally induced laboratory models of inflammation, pyretic activities and also antifungal activity.

 

 

INTRODUCTION:

Most people in the rural areas of the world depend largely on the herbs for the treatment of several ailments because medicinal herbs constitute indispensable components of tradition medicine  practice due to low cost, easy assess and ancestral experience1. Inflammatory responses are mostly associated with pathological disorders 2. Although a good number of plant species are used for this purpose, scientific and pharmacological is scare or very little3. In recent years there has been a growing interest to evaluate plants possessing antimicrobial activities for various diseases4. A number of studies have been reported dealing with antimicrobial screening of extracts of medicinal plants 5,6,7. Plant derived drugs have become a popular alternative medicine in developing countries. Synthetic antifungal / antibacterial drugs widely used at present are sometimes causing toxicity and adverse drug reactions8. Further more, herbal medicines and supplementation are considered less toxic than the synthetic compounds9. However, folk medicines have not been studied extensively.

 


Preparation of extracts (isolated compounds):


 


 

Although several species of solaum are utilized for the cure of various diseases. Several workers reported this activity and the most notable report is activity of solacasine form solanum psuedocapsicum against mycobactamium smigmatitis Mitscher and Wilson. Anti tumor activity of β Solamarine, and Solapalmiotine isolated from S. tripartitum against cells derived from the human carcinoma of nasopharynx (Kupchan) against Sarcoma 180 in mice (cham) were reported Biological activities of α tomatine a glycol alkaloid like antifungal activity, anti microbial and also anti diuretic was reported by Roddick and concluded that activities were exhibited by partially purified methanolic extracts and not purified alkaloid. Roddick has also reviewed some enzymes inhibiting activity of Solanum alkaloids and reported anti cholinesterase activity 10-15

 

Materials and Methods:

Plant material:

The plant has been identified in the Kodaikanal area and has been authenticated by Dr. V. Balasubramanian M.Sc. Phd., Taxonomist, Saraswathi narayana College, Madurai.

 

The plant was collected during months of August-September which is the season for bearing the berries.

 

Since almost all the reports suggest berries to be the main source of alkaloid. We have collected the plant during three stages 1.Berries at green colour stage.2. Berries at greenish yellow 3. Berries at red colour (ripe).

 

Animals:

Wister rats were obtained from the institutional animal house and they were kept in the departmental animal house at 25±2°C and relative humidity 45-51.5%, light and dark cycles of 10 and 14h, respectively for 1 week before and during the experiments for acclimatization. The animals were provided with standard rodent pellet diet and water was allowed ad libitum. Rearing up of animals in the experimental span conformed to the norms of institutional animal Ethical Committee (IAEC), India and ethical guidelines for investigations of experimental pain in conscious animals.

 

Rechromatography:

The fractions CDE (from chloroform: Ethylacetate )and FG (from methanolic chloroform) found to contain alkaloid as T.L.C. they were concentrated and pooled , and dissolved in chloroform. They were rechromatographed on a smaller column using methanol. The alkaloid elude from first four fractions (4x25ml). The methanol was evaporated and alkaloid was crystalised in chloroform in refrigerator for 2hours.


Table 1- Antipyretic activity of various extracts of Solanum leave Dunal

Drug

Initial temp.

 

Temperature in °C after hour shown (SEM ±)

0 hours

1½ hours

3hours

4½hours

Control

37.6

39.2

39.3

39.5

39.5

Standard

37.5

39.2

38.2

37.8

37.6

SA-I

37.6

39.2

39.0

38.6

38.0

SA-II

37.5

39.4

38.6

38.2

37.8

SA-III

37.6

39.2

39.0

38.8

38.2

 

Table 2- Anti-inflammatory activity of various extracts of Solanum leave Dunal

Drug

Dose

mg/kg of body weight

30min

paw volume

%decreaseof paw volume

60min

paw

volume

%decrease of paw volume

120min

paw volume

%decrease of paw volume

180 min paw volume

%decrease of paw volume

Control

Vehicle P.E.G.400

0.62

0.0

0.63

0.0

0.64

0.0

0.66

0.0

Standard drug[indomethacin]

100mg/kg

0.48*

22.58

0.44**

30.15

0.38**

40.62

0.29*

49.82

SA-I

100mg/kg

0.54**

12.90

0.51*

19.04

0.49*

23.43

0.47*

28.78

Sa-II

100mg/kg

0.60*

3.22

0.53*

15.87

0.47*

26.56

0.42**

36.36

SA-III

100mg/kg

0.53**

14.51

0.47**

25.39

0.43**

32.81

0.39**

40.90

*     

 

 

 

 

 

 

 

 

 

 

 

  Not significant, **     Significant, Values are mean ± SEM

 

Table 3- Anti fungal activity of Solanum leave Dunal

Name of the compound

Concentration of sample

Diameter of zone of inhibition

% of zone of inhibition

SA –III

 

 

Control

1%

2%

3%

0.1ml

-

-

5mm

-

-

-

29.4

-

SA –I

 

 

Control

1%

2%

3%

0.1ml

-

-

-

-

-

-

-

-

SA –II

 

 

Control

1%

2%

3%

0.1ml

10mm

12mm

13mm

-

58.8

70.5

76.4

-

 

 

Griseofulvin

1%

2%

3%

10mm

13mm

17mm

58.8

76.4

100

 

 


 

The cholorform extract on drying yielded white powdered with m.p203°C.This compound here after referred SA-I.

 

Anti inflammatory activity:16-20

Carrageenan induced paw odema method:

The crude plant extracts were screened by carrageenan induced rat paw edema method of Winter et al., the extracts, indomethacin standard drug and control vehicle injected   intraperitoneally, 30 minutes prior to the injection of carrageenan.

 

Albino rats of either ex weighing between 150-200gms were randomly distributed and were divided into to five groups, each group consisting of four animals. The volume of paw of each animal was determined before giving any drug. The first group served as control,(P.E.G.400) the second group served as standard, the third group received SA-I, fourth and fifth groups received compounds SA-II,SAIII respectively.

 

All the extracts were dissolved in P.E.G.400 and administered intra peritoneally of the dose of 100mg/kg of body weight of the rats. After 30minutes of duration 0.1 ml of 1% W/V suspension of carrageenan was injected into the sub planter region of left paw of each animal of the five groups.

 

The paw volume was measured at a time interval of 30minutes, 60minutes, 120minutes and 180 minutes after the administration of carrageenan. The degree of edema formation at the hind paw volume was measure by plethsomometer.

 

The volume displacement has been expressed as units are being equivalent to 0.72ml.

 

The percentage inhibition of odema has been calculated by the following formula and results are tabulated and plotted.

  Vc-Vt

--------- X 100

   Vc

Where Vc represents the average increase in paw volume of control and Vt represents the average increase in paw volume after the administration of drug. Table 2

 

Antipyretic activity21-23

Brewer’s yeast method:

Antipyretic activity was conducted on Wistar strain albino healthy male rats weighing about 150-200gms. The rats showing 37.5±0.5°C were selected and then they were fasted for 24 hours before inducing pyrexia. Their normal body temperature was recorded. Pyrexia was induced by injecting subcutaneous by 12% w/v suspension of yeast 1ml/200 body weight and they were allowed to feed. The animals were divided into 5groups of 3 each and numbered.

 

After 10hours rectal temperature was recorded using clinical thermometer introduced 2 cm into the rectum and keeping it inside for one minute. The temperature recorded first after 10 hours of yeast administration was taken as zero hour recording. The control, standard and the compounds were given to the animals by asgric tube. After the drug was administered, the temperature of all rats in each group were recorded at an interval of 1½ hours,3 hours and 4½ hours. The mean temperature was found out for each group and compare with the standard drug values.

 

The animals marked group-I received orally 1ml/200gm of body weight of 0.5% w/v solution of sodium laurylsulphate and served as control.

 

The animals marked group –II received orally 300mg/kg body weight of acetyl salicyclic acid in 0.5% w/v suspension of sodium lauryl sulphate and served as standard the animals marked group-III, IV and V received 300mg/kg of body weight of the various compounds. The results are shown in the graph. Table 1

 

Antifungal activity:24-31

The antifungal activity of the compounds of Solanam leave Dunal were evaluated by cup plate method with test organism pc Candida albicans. The powdered griseofulvin (standard drug was dissolved in sterile water to produce a concentration of 1%,2% and 3% respectively. The solvent control was maintained through out the experiment.

 

Preparation of known concentration of compounds:

Like the standard drug solution, various compounds of the plant were prepared at various concentration of 1%, 2% and 3% solution respectively. The extracts were dissolved in an appropriate solvent.

 

From the solid culture the clinical sample of Candida albicans wee transferred into nutrient broth by using sterile inoculation loop the nutrient broth was incubated for about 24 hours at 37°C. This showed sufficient growth of fungi.

 

Cup-plate or cylinder plate method:

For the screening of antifungal activity pour plate method was used. Petridishes were filled to a depth of 4-5mm with agar medium. Which has been previously inoculated with the test organism. The temperature of the medium shold not exceed above 50°C when the organism were inoculated. The petridishes were placed on horizontal surface to ensure uniform thickness of the media and allowed to solidify.

 

After the agar has solidified 4 holes were made in the medium with a sterile borer. About 0.1ml of 1%.2% and 3% solution of extracts were added to the holes. The compounds were dissolved in the appropriate solvent. A positive control and solvents were also added in the same concentration to another bored petridishes and zone of inhibition was measured after incubation at 37°C for 24hrs. the percentage of zone of inhibition was calculated by using the formula. Table 3

                                               Zone of inhibition of test

Percentage Zone of inhibition = ---------------------------------- X 100

                                                    Zone of inhibition of Std.

 

Statistical analysis:32

All the data were presented as mean ±SEM and analyzed by Dunnett’s test and unpaired Students t-test for the possible significant interrelation between the various groups. A value of p<0.01 was considered statistically significant.

 

Results:

Carrageenan induced paw oedema in rats:

The Solanum leave Dunal at the dose levels of 100mg/kg exhibited resistance against carrageenan induced pain after 30min of the last dose and standard drug. The average percent increase in paw volume indicates pain after treatment was significantally(p<0.01) and dose dependently increased. The significant (p<0.01) effect of standard drug Indomethacin at the dose.

 

Anti pyretic effect:

The result of the antipyretic study showed the subcutaneous administration of the isolated compound SA-I (300mg/kg) caused significant inhibition of the pyrexia induced by yeast. The antipyretic effects of 300mg/kg extracts were comparable to that of acetyl salicylic acid b /w 30 and 60min after treatment.

 

Anti fungal activity:

The compound SA-III(A), SA-I(B),SA-II(C) and the standard drug also prepared various concentrations 1%,2% and 3% and the activity was showed against candida albicans. The percentage of inhibition is significant in SAII(C) as compared to standard drug Griseofulvin.

 

 

Discussion:

This study has investigated the scientific reasons behind the folkloric use of Solanum leave Dunal, in the management of inflammatory conditions, anti pyretic activity. The results indicated that the plant were active against all the experimentally induced laboratory models of inflammation and pyrexia.

 

The isolated  compounds of Solanum leave dunal inhibited the formation of paw oedema to significant levels in rats treated either with Carrageenan  at a dose of 100 mg/kg orally, the SA-III of isolated compound of Solanum leave dunal inhibited the formation of edema to significant levels in rats treated with carrageenan. At a dose of 100mg/kg orally, the SA-III produced 40.90% inhibition in case of the carrageenan induced edema (p<0.01). From that data we can conclude that isolated compound SA-III of Solanum leave dunal inhibits the release of serotonin and bradykinin (2h) and prostaglandin (3h) .As the mechanism of carrageenan is it induces release of histamine (1h), serotonin and bradykinin (2h), prostaglandin (3h).

 

Solanum leave Dunal elicited a dose dependent inhibition of rectal temperature compared with control group. The isolated compound SA-II having maximum antipyretic activity which was compared with standard inhibition at 300mg/kg p.o.

 

The fungal activity of the isolated compounds (SA-I, SA-II, SA-III) were prepared at various concentration of 1%,2% and 3% solution respectively. In this the compound SA-II(C) having significant antifungal activity compared with other compound against Candida albicans.

 

The results indicated that the isolated compounds of Solanum leave dunal were active against all the experimentally induced laboratory models of inflammation, pyrexia and antifungal activities. The Anti inflammatory, antipyretic and antimicrobial activities may be attributed to the presence of alkaloids, carbohydrates, glycosides and steroids in the isolated compounds of  Solanum leave Dunal32-35

 

References:

1.     Marini-Bettolo GB. Present aspects of the use of medicinal plants in traditional medicine. J Ethnopharmacol 1980; 2:5-7.

2.     Akah PS, Nwambie AI. Evaluation of Nigerian traditional medicines: Plants used for rheumatic ( inflammatory ) disorders. J Ethnopharmacol 1994; 4:179-82.

3.     Akah PA, Njike HA. Some pharmacological effects of rhizomes aqueous extract Anchromanese diformi. Fitoterapia 1990; 61:368 -70.

4.     Clark AM, Hufford CD. Discovery and development of novel prototype antibiotics immunodeficiency syndrome, In: Human Medical Agents from Plants, by A Douglas kinghorn and Manuel F Balandrin (Eds), American Chemical Society (ACS Symposium Series 534 ), Washington DC 1993;228-241.

5.     Perumalsamy R, Ignacimuthu S. Antibacterial activity of some folklore medicinal plants used by tribals in Western Ghats of India. J Ethnopharmacol 2000; 69: 63-71.

6.     Portillo A, Vila R, Frexia B,Adzet T, Canigueral S. Antifungal activity of Paraguayan plants used in traditional medicine. J Ethnopharmacol 2001; 76:93-98.

7.     Somchit MS, Mutalib AR, Ruddy HM, Murni A. In vitro antifungal and antibacterial properties of Euphorbia hirta. J Trop Med Plants 2001; 2: 179- 182.

8.     Somchit N, Siti MH and Suraiya HS. Itraconazole and fluconazole-induced toxicity in rat hepatocytes: A comparative In vitro study. Hum Exp Toxicicol 2002; 21: 43-48.

9.     Perry LM. Medicinal plants of East and Southeast Asia: Attributed properties  and uses, MIT Press, Cambridge 1980; 73-81.

10.   Mitscher ,C.,Jvarker J.V.,Experentia .32(4),1976,415.

11.   Wilson R.F., Toxicol appl.pharmacol. 3,1961,39.

12.   Kupchan S.M., Davies A.P., Barboutis S.J., Jr. Org Chem. 34(12),1969,3888-3893.

13.   Kupchan S.M.,Science 150,1965,1827.

14.   Bill E.,Cham., Planta Med.58,1987,34.

15.   Roddick J. Phtochem.13,1974,9.

16.   Telek J. Planta medica ,1997,41,92.

17.   Bill E.,Cham., Planta Med.58,1987,34.

18.   Kulkarani S.K.Handbook of Exp.pharmacy 1st ed.1987,83-84.

19.   Ghosh M.N.Exp.Pharmacology;1984,154.

20.   Robert A. turner screening methods in pharmacologyA.P. 1965.

21.   Pendse VK , Dadich AP, Mathur PN, Bal MS and Madan BR. Indian J Pharmacol.1977;9:221.

22.   Jeyasekar P, Mohan PV  and Rathinam K. Hepatoprotective activity of ethylacetate extract of Acacia catechu . Indian J  Pharmacol. 1997;29: 426.

23.   Loux JJ, Depalma PD and Yankell SL, Antipyretic testing of asprin in rats. Toxicol  Appl Pharmacol. 1972;22:672-673 .

24.   Indian Pharmacopoeia . 1996 VoIIA 100.

25.   Ind.J.of pharma sceince.1997,Vol 59,145-147.

26.   Mackie and Mecartney pra.med.microbiology VoII 15th ed.260.

27.   Bentley’s text book of phar.8th ed. 1997,516-517.

28.   Indian Pharmacopoeia. Microbiological assays and tests.Appendix-9,Vol. II Publication and Information Directorate, India.1996.A-100 to A-116.

29.   Cruickshank R, Duguid JP , Marmion BP, and Swain RHA , Medical Microbiology, Churchill Livingstone, London.1989 pp. 201-208,

30.   Holt JG, Krieg NR, Sneath PH, Stanley JT, and  Williams ST, Bergy’s Manual of determinative Bacteriology, 10th edn (Williams and Wilkins Baltimore).

31.   Pelczar MJ, Chan ECS, and Krieg NR, Microbiology. 5th edn. McGraw – Hill Book Company,  Newyork .1986 .p. 333, 435 and 556.

32.   Amritage P , Eds., In; Stastical Methods in Medical Research, Blackwell Scientific Publications,  London, 1971. p 217 .

33.   Kokate, C. K., Practical pharmacognosy , 4th  edn.(Vallabh Prakashan Pune ) 1996, pp.107.

34.   Harbone ,J.B. Phytochemical methods.1973.

35.   Plaisted ,Philip .H. Contributions from Boyee Thompron Institute1958; 9: pp.231-44. 34.Sethi, P.D., HPTLC Quantitative Analysis of Pharmaceutical Formulations, 1st ed.CBS Publishers and Distributors, NewDelhi. 1996  pp.3-73.

 

 

Received on 11.09.2009

Accepted on 29.11.2009

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Research Journal of Pharmacognosy  and Phytochemistry. 2(1): Jan.-Feb. 2010, 25-29