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Analytical Study of Thalangi Ennai: A Pain Relieving

Siddha Medicated Oil

Vinotha Sanmugarajah, Ira Thabrew,

Sri Ranjani SivapalanUnit of Siddha Medicine, University of Jaffna, Sri Lanka

Institute of Biochemistry, Molecular Biology and Bio technology, University of Colombo, Sri Lanka

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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.

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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 Siddha Agastiyar.

Some of his works are still standard books of Medicine and surgery in daily use among the Siddha Medical practitioners and Traditional healers.

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Siddha Materia Medica The resources of Siddha Materia Medica are classified in to three major divisions namely,HerbalMetals and MineralAnimal 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.

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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.

Siddhars described 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.

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Thalangai ennai 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.

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Botanically pure and authentic ingredients were used in the preparation of T. ennai.

IngredientsIt is a compound medicine and its ingredients are, Juice of the thalangai (Pandanus tectorius) -1250 mlSesame oil - 625 ml Cow’s milk - 625 mlWater - 16000 mlRock salt -10 gSeventeen different herbal raw materials - each 10 g

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Details of ingredients of Thalangai ennaiNo Local name Scientific name Family name Pharmacological

actionPotency

1 Nannari ver Hemidesmus indicus

Asclepiadaceae Analgesic Cold

2 Villamichu ver

Plectranthus zeylanicus

Labiatae Refrigent Cold

3 Vetti ver Andropogon muricatus

Gramineae StimulantRefrigent

Cold

4 Sittamutti Pavonia zeylanica

Malvaceae Analgesic Cold

5 Peramutti ver Pavonia odorata Malvaceae Analgesic Cold6 Tevadaru Cedrus deodara Coniferae Anti

inflammatoryHot

7 Jatamashi Nardostachys jatamansi

Velerianaceae Aromatic Hot

8 Kacholum kaempferia galanga

Zingiberaceae AromaticStimulant

Hot

9 Mara manjal Coscinum fenestratum

Menispermaceae Analgesic Aromatic

Hot and Cold

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10 Sandanam Santalum album Santalaceae Analgesic Cold11 Kottam Costus speciosus Scitaminaceae Stimulant Hot12 Kundurukkan Boswellia serrata Burseraceae Stimulant Hot13 Adi-maduram Glycyrrhizae glabra Papilionaceae Emmolient

AnalgesicCold

14 Satakuppi Peucedanum graveolens

Umbelliferae DeobstruentStimulant

Hot

15 Chirttilam/elakaya

Eletteria cardamomum

Zingiberaceae StimulantAromatic

Hot

16 Patchchilai Zanthochymus piotorlins

StimulantAromatic

Hot

17 Korai-kizangu

Cyperus rotundus Cyperaceae Stimulant Cold

18 Thazhai/ thazaai

Pandanus tectorius(Screw pine/ umbrella tree: Ketaki/ ketaka)

Pandanaceae(Mudu- keyiya)

RefrigentAromaticAntispasmodic

Cold

19 Rock salt NaCl impura Stimulant Hot20 Sesame oil Sesamum indicum Pedaliaceae Analgesic Hot

21 Cow’s milk Bos indicus inn

22 Water

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Preparation Method of T. ennaiFrom 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.

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ObjectiveThe aim of this study was to evaluate the scientific validation of the Thalangai ennai.

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MethodologyFor 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 Thalangai ennai was studied after dissolved the oil in water and extracted with dichloromethane.

The oil sample was purchased from Siddha Drug Unit, Atchuvely, Jaffna.

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Physicochemical evaluationSample of oil (Thalangai ennai) were subjected for determination of physicochemical parameters such as organoleptic characters, pH value and specific gravity.

Organoleptic evalutionThese refer to macroscopic identification of the formulation.Sample of oil (Thalangai ennai), water and dichoromethane extracts of oil were subjected to the organoleptic characterization such as appearance, touch, colour, clarity and odour.

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Determination of pH rangeThe pH of sample of oil (Thalangai ennai) 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).

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Determination of specific 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)

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Development of TLC fingerprints and standardization of thalangai ennai

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 thalangai ennai and fume ships of in an accurately weighed, glass-stoppered round bottom flask.

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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 2 hours 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.

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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.

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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.

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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 extract and heating the plate for 5-10 minute (100-105°C).

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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)

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Results and DiscussionThe 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, Thalangai ennai was subjected to systematic physicochemical and Thin layer chromatography (TLC) analysis to determine the quality and purity of that oil.

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Table No. 1: Analytical parameters of Thalangai Ennai

Parameters Thalangai Ennai

Appearance Viscous

Touch Oily

Colour Golden brown

Clarity Clear

Odour Characteristic

pH value (290C) 4.61

Specific gravity 0.9185

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Table No.2: Organoleptic properties of the water and

dichloromethane extracts of the Thalangai Ennai

Parameters Thalangai EnnaiWater extract Dichloromethane extract

Appearance Liquid Liquid

Touch Liquid Liquid

Colour Light yellow yellow

Clarity Clear Clear

Odour Characteristic Characteristic

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Thalangai Ennai of Government Siddha Drug Unit

Dichloromethane extract of Thalangai ennai

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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.

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TLC finger print analysis of extract of Thalangai Ennai

254 nm 366 nm Visible after spray

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Table 3: TLC finger print analysis of extract of Thalangai Ennai

Solvent system:- Pure Dichloromethane (volume)

Solvent front:- 7 cm

Rf values and colour of spots {*- intense, $- Moderately intense, #- Faint}

0.0286 * Brown 0.4000$ Pink

0.0571$ Pink 0.4428$ Green

0.0714* Brown 0.5286# Pink

0.1143# Grey 0.5857# Green

0.1857# Brown 0.8571# Pink

0.2286$ Pink 0.9143* Orange

0.2571$ Grey 0.9714* Pink

0.3571# Pink

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Table 3, summarizes the Rf values and colour of spots visible in the TLC profiles of the water and dichloromethane extract of Thalangai ennai in 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.

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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.

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ConclusionAs there is not any evidence for detailed physicochemical and TLC evaluation on Thalangai ennai is reported.

Therefore present work is taken up in the view to standardize the Thalangai ennai.

In the present study Thalangai ennai was subjected to physicochemical parameters and TLC finger printing for standardization.

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

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AcknowledgementThe 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.) Menuka Arawwawala, Industrial Technology Institute (ITI), Colombo 07, to conduct the physicochemical studies are gratefully acknowledged.

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THANK YOU

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TLC finger print analysis of extract of Thalangai Ennai

254 nm 366 nm Visible after spray