Analytical Study of Thalangi Ennai : A Pain Relieving Siddha Medicated Oil

1. Analytical Study of ThalangiEnnai: A Pain Relieving Siddha Medicated Oil VinothaSanmugarajah, Ira Thabrew, Sri RanjaniSivapalan Unit of Siddha Medicine, University of Jaffna, Sri Lanka Institute of Biochemistry, Molecular Biology and Bio technology, University of Colombo, Sri Lanka

2. Introduction • Siddha, Ayurveda and Unani are the three major Traditional Systems of Medicine in Sri Lanka. • Siddha which is largely therapeutic in nature has a rich heritage and history. • The word ‘Siddha’ means ‘Perfection’ and those who attained perfection were known as ‘Siddhars’ the pioneers of the system.

3. The Siddhars were great scientists in ancient times. • Siddha is widely practiced in the Tamil speaking areas of Northern and Eastern provinces. • According to tradition, the origin of Siddha system of Medicine is attributed to the great SiddhaAgastiyar. • Some of his works are still standard books of Medicine and surgery in daily use among the Siddha Medical practitioners and Traditional healers.

4. SiddhaMateriaMedica • The resources of SiddhaMateriaMedica are classified in to three major divisions namely, • Herbal • Metals and Mineral • Animal kingdoms • According to Siddha literatures, there are 64 dosage forms of which 32 are meant for oral (internal) route and 32 for parental & external (including topical) applications.

5. Effective management of vātā disorders such as rheumatic pain, bone degenerative changes and impaired movement of limbs are the important tasks in the field of treatment. • Siddharsdescribed vātā disorders of different origin and external application is most useful for their management. • Among them Ennai (Medicated oil) is one of the medicaments prescribed by them.

6. Thalangaiennai is one of the Siddha medicated oil mentioned in the Siddha Pharmacopeia and it is prepared at Siddha Drug Unit in Jaffna and supplied to all Government District Ayurvedic Hospital, Rural Ayurvedic Hospitals and Central Ayurvedic Dispensaries in Jaffna District. This oil is showing clinically enhanced pain relieving properties which requires analytical study for standardization.

7. Botanically pure and authentic ingredients were used in the preparation of T. ennai. Ingredients It is a compound medicine and its ingredients are, Juice of the thalangai (Pandanustectorius) -1250 ml Sesame oil - 625 ml Cow’s milk - 625 ml Water - 16000 ml Rock salt -10 g Seventeen different herbal raw materials - each 10 g

10. Preparation Method of T. ennai From 1 to 17 dried raw materials are ground to get coarse powder and mixed with water. Then continued boiling to reduce the volume to one eighth (2000 ml). Juice of the thalangai, sesame oil, milk (2:1:1) are taken in a vessel and heated for some time; mixed the aqueous decoction together. This mixture is boiled on mild fire with stirring and boiling continued till all the water evaporates and this mixture turn as oily (waxy like). After cooled 10 minutes, the powdered rock salt mixed with the oil.

11. Objective The aim of this study was to evaluate the scientific validation of the Thalangaiennai.

12. Methodology For the scientific validation of this oil, Physicochemical analysis such as pH value, specific gravity and organoleptic characters such as colour, odour, appearance, touch and clarity were performed. Thin Layer Chromatography (TLC) finger print of the Thalangaiennai was studied after dissolved the oil in water and extracted with dichloromethane. The oil sample was purchased from Siddha Drug Unit, Atchuvely, Jaffna.

13. Physicochemical evaluation • Sample of oil (Thalangaiennai) were subjected for determination of physicochemical parameters such as organoleptic characters, pH value and specific gravity. • Organolepticevalution • These refer to macroscopic identification of the formulation. • Sample of oil (Thalangaiennai), water and dichoromethane extracts of oil were subjected to the organoleptic characterization such as appearance, touch, colour, clarity and odour.

14. Determination of pH range • The pH of sample of oil (Thalangaiennai) was determined using standard simple glass electrode pH meter (Consort). The oil was weighed to about 40ml in separate beaker and the pH of the oil was determined using a pH meter at room temperature (29 0C).

15. Determination ofspecific gravity • (http://www.engineeringcivil.com) • Clean, dry and weighed the specific gravity bottle along with the stopper (Weight ‘A’). Then filled the specific gravity bottle with freshly distilled water and inserted the stopper firmly. Kept it in a room temperature and weighed it (Weight ‘B’). Finally, filled the specific gravity bottle with the oil sample and weighed separately (Weight ‘C’). • Specific gravity (oil) = (C – A) • (B – A)

16. Development of TLC fingerprints and standardization of thalangaiennai TLC finger print of the T.ennai was studied after dissolving the oil in water and extracted with dichloromethane. Prior to testing, prepared a water and dichloromethane extract of the oil to examined, used a rapid extraction process, as followed. Place about 50 ml thalangaiennaiand fume ships of in an accurately weighed, glass-stoppered round bottom flask.

17. For the water extraction, 20 ml water was added to the flask and weighed to obtain the total weight including the flask. Then extraction was done by using reflux condenser for 2hours on heating mantle (Electro thermal). Then cooled and transferred to the Separator funnel system. After 20 minutes, the water extract was separated in to the clean small flask and kept tightly closed with lid. Further 20 ml water added to the residue in the round bottom flask and water extraction was done again as the same procedure.

18. After that, the whole separated water extract (30 ml) transferred in to the separator funnel system. Then 10 ml dichloromethane added to the water extract and mixed well. After 20 minutes, the dichloromethane extract was separated in to the accurately weighed, small round bottom flask and kept tightly closed with lid. Further 10 ml dichloromethane added to the residue in the separator funnel system and dichloromethane extract was separated again as the same procedure.

19. Then this procedure repeated again. Totally, dichloromethane extract separation was done for three times. Whole dichloromethane extract collected in to the round bottom flask and evaporated on a Rotary evaporator (Buchi) for just as long as was required to remove the solvent, and re-dissolved the residue in a small volume of dichloromethane (3 ml). Finally, the whole extract was collected in clean stoppered glass test tube and used for spotting the chromatographic plates.

20. Five µl of oil extract was spotted on to TLC plates (8.5 x 5.3 cm) coated with silica gel (pre-coated, GF254) and separated using different solvent systems. • After development visible spots were not found for oil extract. Numbers of spots were observed under UV light (254 & 366 nm). • Visualization was attempted by spraying with vanillin sulphate reagent for oil extractand heating the plate for 5-10 minute (100-105°C).

21. The colour and Rf values of the spots were recorded carefully and the chromatogram was documented by graphical copying. • (Rf value = The distance between the point of application and the centre of the spot of the material being examined / The distance between the point of application and the solvent front)

22. Results and Discussion The physicochemical standards available for the standardization of siddha oils are insufficient. The analytical values available in the pharmacopoeia standards for siddha formulations are not finger print standards for each oil. The first step towards this goal, Thalangaiennai was subjected to systematic physicochemical and Thin layer chromatography (TLC) analysis to determine the quality and purity of that oil.

25. ThalangaiEnnai of Government Siddha Drug Unit Dichloromethane extract of Thalangaiennai

26. According to Table 1, The results showed that this oil (T. ennai) contains specific gravity 0.9185 and pH value 4.61 at room temperature (290C). • As seen in Table 2, both extracts of the oil (T. ennai) had similar organoleptic properties except for the colour of the water and dichloromethane extracts.

27. 254 nm 366 nm Visible after spray • TLC finger print analysis of extract of ThalangaiEnnai

29. Table 3, summarizes the Rf values and colour of spots visible in the TLC profiles of the water and dichloromethane extract of Thalangaiennaiin Pure Dichloromethane solvent system. • Although the separation of the extracts occurred in the solvent system comprised of hexane: dichloromethane: 1% methanol in dichloromethane (1: 4: 5 v/ v) as the mobile phase, the best separation of the extracts occurred in the solvent system comprised of pure dichloromethane (v) as the mobile phase.

30. The TLC of finger print of the oil sample showed fifteen spots with Rf values ranging from 0.0286 to 0.9714 in the pure dichloromethane solvent system.

31. Conclusion As there is not any evidence for detailed physicochemical and TLC evaluation on Thalangaiennaiis reported. Therefore present work is taken up in the view to standardize the Thalangaiennai. In the present study Thalangaiennaiwas subjected to physicochemical parameters and TLC finger printing for standardization.

32. Although, these preliminary physicochemical and the TLC standards presented in this study can be used as finger print standards for Thalangaiennai, further research work of TLC studies of each single ingredient of T.ennai should be carried out in future.

33. Acknowledgement • The financial support provided by the Higher Education Twenty-first Century (HETC) Project, Ministry of Higher Education, Nugegoda, Sri Lanka (Reference No - JFN/ Sidda/ N1) • The guidance given by Dr. (Mrs.) MenukaArawwawala, Industrial Technology Institute (ITI), Colombo 07, to conduct the physicochemical studies are gratefully acknowledged.

34. Thank you

36. 254 nm 366 nm Visible after spray • TLC finger print analysis of extract of ThalangaiEnnai