Studies on Antimicrobial and Anti-inflammatory activity of the Siddha Formulation (Thailam – Medicated oil)

R Badmanaban*¹, CN Patel², P Devi², DJ Sen¹ KM Modh¹

¹Shri Sarvajanik Pharmacy College, Near Arvind Baug, Mehsana-384001, North Gujarat, India

²K.M College of Pharmacy, Uthangudi, Melur Road, Madurai, Tamilnadu-625107, India

ABSTRACT:

Pongamia pinnata and Boerhaavia diffusa are well-known plants and a weed respectively plays in Indian traditional system of medicine. On the basis of its traditional use and literature references, these herbal plants are undertaken in a view to formulate milder and safer herbal topical formulations. It is prepared in the form of “Thailam” using sesame oil as base for bringing about the anti-inflammatory and antimicrobial drugs. To satisfy the desired characteristic of an ideal herbal formulation and also to prove its therapeutic potency, the following parameters like physical, chemical and biological evaluation have undertaken to fix the quality. For the antimicrobial studies strains used like S. aureus, B. substilis (Gram +ve), E. coli, P. aeruginosa (Gram-ve) and Candida albicans, Aspergillus niger (Fungi) were used. Antibacterial activity and antifungal activity of the formulated oil was comparatively lesser than that of the standard drug but formulated oil is significantly more than that of sesame oil base (**P<0.01).By using Carrageenin induced hind paw edema method the Anti inflammatory activity was employed and it was noticed that the Thailam had lesser activity than the standard Diclofenac sodium gel (**P<0.01). But significantly more than that of sesame oil base (*P < 0.05), the overall results revealed that it has effectiveness. In conclusion that the formulated oil in the form of Thailam as its own significant properties, hence it can be used as a safer formulation in near future

KEY WORDS: Formulated oil (Thailam), Sesame oil (Base), Carrageenin, Antimicrobial strains.

INTRODUCTION:

More complex formulation containing several plants parts and in some time with very similar chemical and pharmacological property. In the absence of suitable and simple methods of analysis for complex formulations, the complete analysis may be impractical and only choice left to the control chemist is that he/she analyzes the raw material and then personally check that the desired ingredients are blended as directed in the formula card. In some cases though the chemical nature of the active constituent is not known or it is not possible to isolate without changing its chemical integrity then the only way left to the quality assurance chemist is to standardize it biologically. Boerhaavia diffusa, Linn, Nyctaginaceae family, Chemical Constituents contains crystalline acid. Root contains alkaloid punarnavine, C-methylflavone, rotenoid analogues – boervinone A, B, C, D, E, F, punarnavoride, amino acids, fatty acids, hentriacontanes, b-sitosterol. The root is well-known for its diuretic properties. Taken in large doses it acts as an emetic. It is used in jaundice, ascites, anasarca, scanty urine and internal inflammations. Mixed with dried ginger, it is given in urticaria. Pongamia pinnata (Linn) belonging to the family, Papilionaceae.Chemical Constituents¹ of Seeds contain glabrachromene II, B-sitosterol karanjin, pongamol, pongaglabrone and pongapin, a furoflavone and karanjone.

Seed oil contains furanoflavones – demethoxy Kanugin – isolonchocarpin, chromenochalcone – glabrachalone. The seed is bitter and acrid; carminative, purifies and enriches the blood relieves inflammation, cures earache, lumbago, chest complaints, chronic fevers, hydrocele. The oil is styptic, anthelmintic, and good in scabies, leprosy, piles, ulcers, lumbago, chronic fevers and pain in the liver. The ash strengthens the teeth (Unani).The seeds are used as an external application in skin diseases. The expressed oil of the seeds is used in these diseases as well as in rheumatism²Sesamum indicum, Linnbelonging to the family Pedaliaceae, Leaves contain flavanoid glucoside – pedalin seeds contain furfuran lignans-sesamolin and sesangolin, antioxidant – sesamolinol, sesamol and g-tocopherol. Seeds yield oil containing sesamin and sesamolin³ The seeds are acrid with a sharp bitter sweet taste; oleagenous, indigestible; tonic, cooling, galactagogue, diuretic, astringent to the bowels, aphrodisiac, promote the growth of hair; useful in diarrhoea, gouty joints, urinary concretions, eye diseases; applied to ulcers and piles; cause “Kapha” and biliousness (Ayurvedha).⁴

MATERIAL AND METHODS:

CRITICAL EVALUATION OF SESAME OIL AND FORMULATED OIL:^5,6

Phytochemical evaluation:⁷

The sesame oil and formulated oil (Thailam)⁸ was prepared and it was subjected to hydrolysis with 6N hydrochloric acid for 48 hours (there by the peptide bonds present in the proteins will get break out). Then the hydrolyzed oils were subjected to the following test:

Detection of Protein and Amino Acids:

Biuret test, Ninhydrin Test, By Whatman Filter Paper Test and also by TLC

Thin layer Chromatography Patterns

Solvent System - n-butanol, glacial acetic acid, water (4:1:5).

Spraying reagent -0.2% w/v Ninhydrin solution

Detection of Glycosides:

Legal’s Test, Borntrager’s Test

Hydrolysate was dissolved in pyridine and sodium nitro prusside solution was added to this and then made alkaline with sodium hydroxide solution.

Detection of Phytosterols:

The sesame oil and the formulated oil were refluxed with a solution of 0.5N alcoholic KOH for six hours on a water bath for completion of Saponification. Then the residue was diluted with water. The aqueous mixture was extracted with solvent ether in thrice .The combined ether extract was washed with water and the aqueous washings were rejected, the ether extract was dried over anhydrous sodium sulfate. Residue was subjected to Salkowski and Liebermann – Burchard’s test.

PHYSICAL PARAMETERS OF THE OILS:

The oils were subjected to determine the various physical parameters viz: Physical appearance, solubility, (Table 1 and 2) Determination of PH, specific gravity and refractive index.

Determination of PH:

PH of the oils was determined by using the (Digisun Electronics) by immersing the electrode to a depth of 1cm. The averages of triplicate observations were recorded.

Table 1: Physical Appearance

Parameters	Sesame oil	Formulated oil
Colour	a clear, light yellow liquid	a dark yellow liquid
Odour	Slight sesame seed	Pongamia seed odour
Taste	acrid with a sharp bitter taste	bitter taste
Visual appearance	oily liquid	oily liquid
Consistency	easily spreadable	easily spreadable
Feel on application	smooth and good	smooth and good

Table 2: Solubility Test

Solvents	Sesame oil	Formulated oil
Alcohol	slightly soluble	slightly soluble
Ether	miscible	miscible
Chloroform	miscible	miscible
Hexane	completely miscible	completely miscible
Carbon disulphide	completely miscible	completely miscible

Specific Gravity Test:

Specific gravity is important criteria of the quality and purity of a fixed oil. It may be defined as the ratio of the weight of a given volume of oil at 20°C to the weight of equal water at 20°C. (Table 3)

Table 3: Specific Gravity Test

Samples	Specific gravity
	Values	Standard values
Sesame oil	0.919 ± 0.02	0.916 – 0.921
Formulated oil	0.925 ± 0.01	0.925-0.945(Lab optimized)

Refractive Index:

When a ray of light passes from a less dense to a denser media, it is bent or refracted towards then normal, if ‘e’ represents the angle of refraction and I represents the angle of incidence, according to the law of refraction.

Sin I N

------- = ------

Sin e n

Where n is the index of refraction of the less dense medium and N is the index of refraction of the denser medium. Refractometers offer a rapid and convenient method for the determination of the physical constants. The Abbe type is used for the routine analysis of fixed oils.(Table 4)

Table 4: Refractive Index

Samples	Refractive index
	Values	Standard values
Sesame oil	1.4651 ± 0.02	1.4650 – 1.4665
Formulated oil	1.6424 ±0.02	1.6424-1.6468(Lab optimized )

Mean ± S.D (n=3)

CHEMICAL PARAMETERS OF THE OILS:

Qualitative Chemical Test:⁹

The procedures for qualitative chemical tests have been taken from the standard book (IP – 1996). The values are given in the (Table 5)

Chemical test for sesame oil ^10,11

The characteristic phenolic component is the bases of the BP test for identify and also the test for the detection of sesame oil in other oils. The original test involved the production of a pink colour when the oil was shaken with half its volume of concentrated hydrochloric acid containing 1% of sucrose (Baudouin’s test). However, some commercially refined oils may not give a positive Baudouin’s test. With the current BP test the reagents are acetic anhydride, a solution of furfuraldehyde and sulphuric acid was used for testing.

Table 5: Qualitative Chemical Test

Parameters	Sesame oil	Formulated oil	Standard(Base used)
Saponification value	191 ± 2.22	198 ± 2.56	188-195
Acid Value	2ml ± 0.41	2.3ml ±0.52	Not more than 2ml
Ester value	189 ± 2.14	195.7 ±2.35	186-193
Acetyl value	8.4 ±1.02	9.4 ±1.05	1.1 – 9.8
Iodine value	108 ±2.15	116 ±2.36	103-116
Unsaponifiable matter	1.5 % ±0.18	2.0 % ±0.56	Not more than 1.5 %

Mean ± S.D (n=3)

Quantitative analysis of Heavy metals by UV-Visible Spectroscopy method:¹²

The metal analysis has been carried out at Textile Committee (Ministry of Textile, Govt. of India, Madurai district, Tamilnadu.) in Textile Testing Services – Eco Cell laboratory. Test for metals were carried out for Cadmium, Nickel, Zinc, Iron and Copper by UV-Visible Spectroscopy method (in ppm) level of detection. Results were shown in the (Table 6).

Table 6: UV-Visible Spectroscopy method (in ppm)

Metals	Sesame oil	Formulated oil
Cadmium (ppm)	Nil	Nil
Nickel (ppm)	Nil	Nil
Zinc (ppm)	Nil	Nil
Iron. (ppm)	Nil	Nil
Copper (ppm)	Nil	Nil

ACCELERATED STABILITY STUDIES:¹³

The accelerated stability studies for the formulations are very important in assessing the ability of the products in resisting deterioration. The temperature conductors selected for stability studies represented the extreme exposure conditions that the formulation may have to undergo during storage and usage period. The formulations were filled in suitable containers. All the formulations were stored at different temperatures 4°C, 31°C, 45°C and 60°C in a thermostatically controlled oven. They were observed for color, odour and appearance. (Table 7 and 8)

Table 7: Accelerated stability studies of Sesame Oil

Sr. No	Duration	Temperature
		4°C	31°C	45°C	60°C
1.	1 Hour	+	-	-	-
2.	4 Hour	+	-	-	-
3.	24 Hour	+	-	-	-
4.	48 Hour	+	-	-	+
5.	1 Week	+	-	-	+

Note: (-) indicates no physical change, (+) indicates physical change (odour, colour, turbidity and solidify)

PRIMARY SKIN IRRITATION TEST:¹⁴

The skin irritating test was performed for the formulation in Guinea pigs employing three groups each containing three animals. Two groups were received oils, which was placed on a piece of a cotton wool and it was placed firmly on the pre-shaved portion of the dorsal skin with the help of adhesive plaster. One group was kept as negative control (cotton wool soaked in 0.8% formalin was used as standard to induce irritation). Development of edema and erythema (if any) in the animals were observed for 3 days.

Table 8: Accelerated stability studies of Formulated oil

Sr. No	Duration	Temperature
		4°C	31°C	45°C	60°C
1.	1 Hour	+	-	-	-
2.	4 Hour	+	-	-	-
3.	24 Hour	+	-	-	-
4.	48 Hour	+	-	-	+
5.	1 Week	+	-	-	+

Note: (-) indicates no physical change; (+) indicates physical change (odour, colour, turbidity and solidify)

ANTI – INFLAMMATORY ACTIVITY OF FORMULATED OIL AND SESAME OIL:^15-18

Among the many methods used for screening of anti-inflammatory drugs one of the most commonly employed techniques is based upon the ability of such agents to inhibit the edema produced in the hind paw of the rats after injection of a phlogistic agent (irritant), like Carrageenin, the effect can be measured in several ways. Usually, the volume of the paw is measured before and after application of the irritant and the paw volume of the treated animals is compared to the controls.

Materials Required:

Albino rats, Carrageenin (1% w/w), Diclofenac sodium gel, Sesame oil, Formulated oil Plethysmograph, Normal saline

Carrageenin Induced Hind Paw Edema:

Method:

Albino rats of either sex weighing from 150-200gms were divided into 4 groups and 5 animals in each group. The dosages of the drugs administered to different groups were as follows:

I - Control group (B.P Simple ointment) only base

II - Standard Group (Diclofenac Sodium Gel) for topical application

III - Group receiving formulated oil for topical application

IV - Group receiving Sesame oil for topical application

Both the test samples and STD drug were given externally to respective animals according to body weight 30 minutes before the commencement of the study. After that 0.1ml w/v Carrageenin solution in normal saline was injected into sub plantar region of the left hind paw for evaluation and right hind paw-served as the reference control. The volume of mercury displaced in the Plethysmograph was measured at 0hour, 1^st hour, 2^nd hour, 3^rd hour, 4^th hour and 5^th hour. The percentage decrease in paw edema of the treated group was compared with that of control and the inhibitory effects of the oils were studied. The relative potency of the oil under investigation was calculated, based upon the percentage of inhibition of inflammation. The volume displacement has been expressed as units, one unit being equivalent to 0.072ml. The results have given the (Table 9) and (Graph 1-2)

Table 9: Anti-inflammatory activity of formulated oil and sesame oil

Animal Groups	Paw volume in ml ± S.E.M and its % of inhibition
	0 Hour	1^st hour	2^nd Hour	3^rd Hour	4^th Hour	5^th Hour
Control	0.36 ± 0.01	0.55 ± 0.09 NS	0.76 ± 0.03 NS	0.84 ± 0.05	0.91 ± 0.05	0.93± 0.03
Standard (diclofenac sodium gel)	0.35 ± 0.04	0.37 ± 0.08 NS 32.72 %	0.39 ± 0.09** 48.68 %	0.38 ± 0.02** 54.76 %	0.36± 0.05** 60.34 %	0.37 ± 0.04** 60.21 %
Formulated oil(Thailam)	0.36 ± 0.01	0.47 ± 0.03 NS 14.54 %	0.58 ± 0.09 NS 23.68 %	0.62 ± 0.04** 26.19 %	0.60 ± 0.07** 34.06 %	0.59 ± 0.09** 36.55 %
Sesame oil (Base)	0.35 ± 0.05	0.50 ± 0.04 NS 9.09 %	0.65 ± 0.08 NS 14.47 %	0.72 ± 0.02 NS 14.28 %	0.70 ± 0.06 NS 23.07 %	0.72 ± 0.03* 22.58 %

Values are expressed as mean ±SEM. (n=6) **P<0.01, *P < 0.05 considered significant changes occurred when compared to the control group. NS-indicates that no significant changes in the result.

Graph 2: Anti-inflammatory activity – % Inhibition of paw volume

C - T

% inhibition= -------- X 100

C

C - Increase in paw volume of control

T - Increase in paw volume of test

ANTI-MICROBIAL ACTIVITY STUDY: ^19-21

The Petri dishes were cleaned, dried and sterilized. Then they were filled with a Nutrient Agar or Sabouraud’s Dextrose agar medium with uniform thickness. After solidifying, the plates were inoculated with the organisms of Gram positive, Gram negative bacteria and fungi (Procured from microbiological lab). Three holes were made in the inoculated plates by means of aluminum or stainless steel sterile borer, with the height of 10 cm and 6-8 mm internal diameter. In each hole, the each oils and Ciprofloxacillin, Griseofulvin standard must be added under aseptic conditions. The oils and the standard were dissolved in dimethyl sulphoxide (DMSO) and the 0.1 ml of was introduced into the cylindrical hole by means of micropipette which has (1:5) ml ratio conc. of respective extracts and 100 mcg /ml conc. of standards were added to the holes. Then the plates were kept in the refrigerator for two hours for diffusion. All the Petri dishes were incubated at 37ºC for 24 hours for bacteria and 25ºC for 48 hours for fungal strains .The zone of inhibition was measured by using antibiotic zone reader. The results have given the (Table 10 and 11).

Table 10: Anti bacterial activity by cup-plate method

Sr. No	Bacterial (Strains)	Zone of inhibition in mm
		Standard	Test Samples
		Ciprofloxacillin	Sesame oil	Formulated oil
1.	S.aureus (Gram+ve)	24	10	18
2	B.substilis (Gram+ve)	18	9	12
3.	E.coli (Gram-ve)	22	11	17
4.	P.aeruginosa (Gram-ve)	20	10	14

DMSO as such did not shown any activity

Table 11: Anti fungal activity by cup-plate method

Sr. No	Fungi (Strains)	Zone of inhibition in mm
		Standard	Test Samples
		Griseofulvin	Sesame oil	Formulated oil
1.	C.albicans	19	9	13
2.	A.niger	22	11	16

DMSO as such did not shown any activity

Statistical analysis:

The results are expressed as Mean ± SEM. Data was evaluated using one-way ANOVA followed by Dunnett’s tests. Probability values less than (p< 0.05) were considered significant by using Graph pad Prism 4 version.

RESULTS AND DISCUSSION:

For the Detection of Protein and Amino Acids, both the oils showed the light violet colour, Intense pink spots appeared in the hydrolyzed sesame oil as well as formulated oil showed (Table 12)Thin layer Chromatography Pattern RF values (0.2,0.45,0.6),The saponified oils were dissolved in chloroform, to this add Liebermann’s – Bur chard’s reagent. Greenish colour was produced in formulated oil but not in sesame oil. Hydrolysate was treated with chloroform, and then chloroform layer was separated, and equal quantity of dil. Ammonia was added. The ammonical layer shows the pink in formulated oil and in sesame oil were shown in (Table 13).Phytochemical evaluation of the sesame oil and formulated oil showed the presence of proteins and amino acids, steroids and glycosides.

In physical and chemical method of standardization the tests applicable for fixed oils were carried out. The oils were subjected to determine the various physical parameters Viz: Physical appearance, solubility, PH was 7 for both the sesame and formulated oils. Specific gravity and refractive index

Table 12: Detection of proteins and amino acids

Table 13: Detection of Glycosides

Sr. No.	Test	Observation	Inference
1.	Borntrager’s test: 2 ml of filtrate hydrolysate + 3 ml of chloroform à shaken, chloroform layer was separated and 10 % NH3 solution was added to it.	Formation of pink colour	Presence of glycosides
2.	Legal test: About 50 mg extract dissolved in pyridine. Sodium nitropruside solution was added and made alkaline using 10%NaOH solution.	Characteristic pink colour	Presence of glycosides

Sr. No.	Test	Observation	Inference
1.	Millon’s test: 2 ml of filtrate + few drops of Millon’s reagent.	White precipitate	Presence of protein
2.	Biuret test: 2 ml of filtrate + 1 drop of 2% CuSO4 + 1 ml of 95% ethanol + excess of KOH pellets.	Pink colour in the ethanolic layer	Presence of protein
3.	Ninhydrin test: 2 drops of ninhydrin solution. + 2 ml of aq. filtrate.	Characteristic purple colour	Presence of amino acid

Since the oil was prepared by boiling with plant parts as well in a metal container at 200 ºC, the presence of heavy metals were anticipated. Heavy metals analysis was performed by UV-Visible Spectroscopy method (in ppm) level of detection. Results were shown absent for all the metals which we tried out to detect for safer formulation for skin disorders. Accelerated Stability Studies result at 4°C; both sesame oil and formulated oil were solidified. At 31°C, no changes occurred. When the product was kept for about 1 week at 45°C there was a change in colour, odour and appearance. When the temperature was raised to 60°C and the time of exposure was prolonged to about 48hours and 1 week the product showed the same changes. So, the stability of the product depends on the temperature and time.

Antibacterial and antifungal activities were done using Gram positive organisms of S.aureus, B.substilis and Gram Negative organisms of E.coli, P.aeruginosa and Candida albicans, Aspergillus niger strains both the antibacterial and Antifungal activity was comparatively lesser than that of the standard drug but significantly more than that of sesame oil base. (Graph 3 and 4)

Graph 4: Results of Antifungal studies

Anti inflammatory activity using Carrageenin induced hind paw edema method was employed and it was noticed that the Thailam had lesser activity than the standard diclofenac sodium gel. But significantly more than that of sesame oil base, the overall results revealed that it has some kind of effectiveness.

The formulation was tested for skin irritating using Guinea pig model. For the formulated oil and sesame oil there was no development of edema and erythema. No reaction was noticed. It was found to be safe.

CONCLUSION:

Thailam, a Siddha formulation was developed using two plant drugs namely Pongamia pinnata seeds and Boerhaavia diffusa roots. Critical examinations of these drugs were carried out to fix out the authenticity. Thailam was prepared by boiling the powdered drugs with sesame oil which is acting as menstrum or base. Then the formulated oil and sesame oil were subjected to different quality parameters like physical, chemical, spectroscopical, microbiological and pharmacological aspects. So we may conclude that the formulated oil in the form Thailam as its own significant properties, hence it can be used as a safer formulation in near future.

ACKNOWLEDGMENT:

I express my profound sense of gratitude my Principal, K.M. College of Pharmacy, Madurai, Tamilnadu, for provided me the permission to utilize the facilities of the Pharmacology laboratory to carry out my postgraduate project work.

REFERENCES:

1. Chelladurai.V. Medicinal plants of India, 2000, (2) 80:pp437, 493.

2. Kiritikar, K.R. and Basu,B.D, Indian Medicinal Plants, 1987, I:pp830-832, III:pp1857-1861, IV:pp2044-2048.

3. The wealth of India, publications and Information Directorate (CSIR), New Delhi 1972, II: pp174-176, VIII: pp204-210, IX:pp 278-281.

4. Flora of the Gamble J.S., Presidency of Madras, Published under the authority of the Secretary of State for India in Council, 1935, I, II, III.

5. Indian Pharmacopoeia, Pub. Govt. of India of Ministry of Health 1996, II, A-50, A-51, A-52, A-53, A-54, A-124, A-74.

6. World Health Organization, Geneva, Quality Control methods for Medicinal plant materials, 1998:pp28, 30.

7. Kokate.C.R., Practical Pharmacognosy, Pub, valiabh Prakasham, Delhi, 4^th Edition 1994:pp108, 178-181.

8. Badmanaban. R, Patel C.N and Devi. P, Physio-chemical investigation and wound healing activity of Sesame oil and formulated oil ,Asian J. Research Chem. 2009; 2(4):501-505

9. Trease and Evan’s, 14^th Edition, 1997:pp179, 185,180.

10. British Pharmacopoeia, 1993, I: pp594.

11. Indian Pharmacopoeia, pub. Govt. of India of Ministry of Health, 1966:pp640, 649-650.

12. Remington – The science and practice of Pharmacy, 14^th edition, 1920, I; pp965-970.

13. Leo Lakhman, Herbert.A. Liberman, Kanig.T. The Theory and Practical of Industrial Pharmacy, 3^rd Edition, 1987:pp314, 296-302, 487,190-193.

14. Satoshkar.R.S. Bhandarkar,S.D., Ainapure.S.S., 2001, 17^th Edition, 154:pp835.

15. Winter C.A, Rishley A.Nuss Gow, Soci. Exp. Biology, 1962, 3: 544.

16. Singh H and Gosh M.N., J. Pharm, Pharmacology, 1968, 20:316.

17. Kelley.W.B and Weiner, Text book of Rheumetology, Sanders Company, Philodelphia, 1981:pp1018.

18. Armitage P, In. Statistical method in Medical Research, Blackwell Scientific publication, 1971: pp 217.

19. Rathee.P.S., Mishra.S.H. and Kaushal.R., Indian Drugs, 1979, 16(9):207.

20. Rathee. P.S., Mishra.S.H. and Kaushal.K., Indian Drugs, 1980, 17(5):136.

21. Banerjee.A., and Nigam.S.S., Indian Drugs, 1980, 17(7):212.

Received on 04.12.2009

Accepted on 16.01.2010

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Research J. Pharmacology and Pharmacodynamics 2(1): Jan. –Feb. 2010: 83-88