Pharmacognostic Evaluation and Analgesic Efficacy of Ethanolic Extract of Euphorbia dracunculoides

Background: Euphorbia dracunculoides L is available in market in raw form. It is used by people for the treatment of warts, snake bite and epilepsy. Present study is about Euphorbia dracunculoides L. belonging to the family Euphorbiaceae, comprises pharmacognostic study, physiochemical analysis and their pharmacological efficacy. Materials and Methods: The macroscopic study was carried out through sensory organs like size, shape, texture etc. Physiochemical analysis was carried out through proper procedure from relevant literature, and analgesic activity was done through proper method by following literature. Results: Macroscopic study showed that the plant is an annual herb, stem is branched from the base, yellow green at the bottom and light green at the top, glabrous, smooth, contains white latex, its leaves are sessile, alternate, dark green, simple, stipulated, sub-acute or acute at the apex and entire margin. Root is yellowish in colour, conical in shape, smooth texture, downward in position. Powder drug study which was performed revealed various structures. Phytochemical screening includes both qualitative and quantitative analysis which was carried out indicated the presence of carbohydrates, proteins, saponins, sterols, alkaloids, phenolic compounds, glycosides, flavonoids and tannins. Proximate analysis showed proteins, crude fat, crude fibre, carbohydrates, moisture contents and ash. Elemental analysis revealed the presence of macro and micronutrients i-e Na, Zn, Mg and Fe, Cu, Ag and Au. Analgesic effect was dose dependent. Plant extract showed maximum inhibition of writhing 1.66±0.32 (96.61%) at 300 μg/ml. Conclusion: The pharmacognostic study, physiochemical analysis and their pharmacological efficacy is helpful in the standardization of drug.


INTRODUCTION
Pharmacognosy signifies the importance of medicinal plants as cost-effective health factor of biodiversity and also their preservation and sustainable use (Rahman  et al., 2004).The knowledge of certain plants and their unique qualities has been passed on through the generations to ultimately reach us. 1 Natural products derived from plants have been traditionally important in prevention of illness and homeopathic treatment.They are also contained active ingredients for the production of health care products. 2Medicinal plants contain some bioactive organic compounds i-e carbohydrates, steroids, tannins, flavonoids, alkaloids and terpenoides, which provide specific physiological action on the human body.4] Medicinal plants possess enormous potential as beneficial drugs for human beings.Certain chronic and infectious diseases can be effectively treated using the substances present in these plants. 5However, lack of authentic documents and strict quality control has been detrimental to the nervous system.Unlike anesthetics, which eliminate sensation, analgesics simply ease pain in the host.Traditional ethno-medicine used to relieve pain is a potential source for discovering novel anti-inflammatory, antipyretic and analgesic drugs.Natural products are a rich source of chemical substances with beneficial therapeutic capabilities. 10

Collection and preservation
Euphorbia dracunculoides was collected from the southern districts of Khyber Pakhtunkhwa, cleaned and jumbled thoroughly, dried in shade.A specimen was mounted in kept on a herbarium sheet, given a voucher number and kept in the department of Botany.It's macroscopic and microscopic characteristics were studied.An electric grinder was used to thrash the dried plants into powder and then stored in an air-tight bottle that protects it from hostile climate, insect attacks, molds and moisture.Physiochemical analysis is also carried out on a part of this powder and biological studies are performed on the rest.

Morphological observation Macroscopic Study
The macroscopic observation of Euphorbia dracunculoides was carried out by organoleptic method, we analyzed the shape, size, odor, color, petiole, phylotaxis, lamina, surface, taste, Apex, Type, leaf margins, stipule and venation of the leaf, also analyzed colour, habit, taste, odor, size, surface, shape, branching, position, latex, texture, surface in stem, In root we analyzed size, shape, odor, taste, color, position, texture, surface and rootlets. 11

Microscopic Study Powder microscopy
The organoleptic characteristics of Euphorbia dracunculoides in powder form were studied such as microscopial characteristic odor, color and taste.Following Wallis, (2005), who carried out powder drug study.

Procedure
Whole plant shade dried at 25°C for 7 days and grounded using electric grinder.Powders of dried parts were used for the observation of the microscopic characters.A pinch of fine powder was taken on a glass slide treated with chloral hydrate solution and iodine solution, then observed under a microscope using different objective lenses for observation of different anatomical structures, which were then photographed.

Elemental analysis
Elemental analysis of powder from the selected parts was carried out with atomic absorption spectrophotometry for the following trace and heavy metals. 12

Preliminary qualitative phytochemical analysis
Phytochemical analysis was done by following [13][14][15][16][17] Test for reducing sugars Killere killiani test for steroidal Glycosides • Glacial acetic acid was used to separately treat 4 ml of extract along with 5 % FeCl 3 and a few drops of concentrated H 2 SO 4 .A bluish green color appeared in the upper layer and a reddish brown appeared at the seam of the two liquids which confirmed the presence of steroidal glycosides.

Confirmative test for tannins
• Ferric chloride (FeCl 3 ) test: A few drops of FeCl 3 solutions were added separately to extract solution of the selected plant.The color transition from dark green to blue-black indicated the presence of Phenol heads, while the complete blue color meant hydrolysable tannin.The presence of condensed tannins in the test samples was confirmed by the green color.

Detection of Alkaloid
Hydrochloric acid was used to dissolve the ethanolic extracts separately, which were then filtered, and the filtrates were used for different tests for Alkaloid detection.
Dragondroff 's test: A red precipitate was observed once the filtrate was treated with Dragondroff 's reagent, which proved the presence of Alkaloids.
Hager's test: Yellow precipitation occurred once the Filtrates were treated with saturated picric acid solution, which is typical for Alkaloids.

Wagner's test:
A reddish brown color appeared once the filtrates were treated with iodine in Potassium Iodide solution (Wagner's reagent), hence proving Alkaloid presence.

Saponins confirmation
Froth test: Distilled water was used to dilute the ethanolic extracts of the plant.A froth appears once they were vigorously shaken for more than five minutes, thus proving the presence of Saponins.

Detection of Triterpenes and Phytosterols
Salkowski's test: Chloroform was used to dissolve the extracts and the solution was then filtered.A few drops of concentrated H 2 SO 4 were added to the filtrate, the solution was shaken and then kept still.Triterpenes were indicated by a yellow color while phytosterols were indicated by a red color in the lower layer.

Detection test proteins & aminoacids
Xanthoproteic test: A few drops of concentrated HNO 3 were added to 2 ml of extract solution.A yellow color appears once it is heated.The color soon turns orange.

Biuret test
• 2 ml of Biuret reagent was added to the test sample and a violet color is observed which represents the presence of protein.

• Ninhydrin test:
To the plant extract solution 0.2% Ninhydrin solution was added resulted appearance of violet coloration which indicated the presence of protein.

Detection of Oil Spot test
• A tiny amount of powdered drug was pressed between the folds of the filter paper which resulted in a translucent oily stain which represents the presence of fixed oil and fats.

Procedure
In this process, 75 ml of distilled water and 10%KOH were added to 2 grams of extract.The process changed the chlorophylls contents into chlorophyllins (water soluble salt).It was then treated thrice with petroleum ether (75ml) in a separating funnel.First, the flask was weighed (W1), ether portion was collected in the flask and then it was placed in a water bath.When it became concentrated and dried, the flask was placed in a desiccator for cooling.The flask was weighed again (W2) and % sterol was calculated using the following formula

Quantitative chemical analysis
Sterols, Saponins, Tannins and Alkaloids can be determined by quantitative analysis of the whole plant using standard method of 16-18

Alkaloids determination Materials required
Ethanolic extracts, acetic acid, filter paper, beaker, balance, water bath

Procedure
Harborne method 16 was used to determine the quantity of Alkaloids in whole plant extract.100 ml of 10 % acetic acid was added to 2 grams of ethanolic extract in a beaker, which was then covered and kept for four hours.The mixture was then concentrated to one-fourth of its original volume using a water bath.A precipitate formed once concentrated ammonium hydroxide was added drop by drop to the solution.Pre weighted (W1) Whitman filter paper was used to collect the precipitate.Dilute ammonium hydroxide was used to wash it.The filter paper containing the residue was dried and weighed (W2).The amount of Alkaloid was calculated as such: Where X= Weight of alkaloids = W2 -W1 W1= Weight of filter paper W2 = Weight of filter paper + ppt

Procedure
Obadoni and Ochuko 18 method was used to determine the quantity of Saponin.20 ml of distilled water was used to dissolve 2 grams of an ethanolic extract of Euphorbia dracunculoides, which was then moved to a 250 milliliter separating funnel.The mixture was shaken vigorously after a further addition of 20 ml diethyl ether.After draining out the layer of ether, 60 ml of n-butanol was mixed with the aqueous layer which resulted in the precipitation of saponin.Pre weighted (W1) Whitman filter paper was used to collect the Saponin, washed thoroughly with 10 ml of 5 % aqueous NaCl solution.An oven at 40 °C was used to dry the precipitate to constant weight (W2).The Saponin content and the initial weight of the sample were measured and calculated as given below: Amount of saponins (mg/g) = x Weight of sample

Nutritional analysis Determination of Moisture Apparatus
Desiccator, Electric oven, Electric balance, petri dish with lid.Procedure 2 gram of powder was taken in petri plate then weighted it (W1).The petri plate was covered with lid partially; the petri plate was then placed in oven (105C o ) for 4 to 6 hours in order to remove moisture.Then place the petri dish in desiccator for cooling for 30 minutes.After that, weight the petri plate again. 19lculation

Determination of Ash Equipment's required
Desiccator, burner, Muffle furnace, Blow pipe, Crucible, Electric balance, Procedure 2 gram of sample powder was taken in a sterilized crucible (W1).Before placing in muffle furnace it was first burnt with burner with the help of blow pipe.Then place it in furnace (550 o C) for many hours.The furnace was then turned off and the color of sample changed into grayish white.Then the crucible was transferred into dessicator in order to cool.Weight it again (W2).The % Ash was calculated by using the following formula. 20ndicator i.

Apparatus
Volumetric flask, pipette, Kjeldahl flask, burette, digestion and distillation apparatus etc. Procedure 0.3g of the powder sample was taken in KJELDAHL flask.7 ml of conc.H 2 SO 4 and 1.5 g of catalyst were added to the flask.It was mixed thoroughly by spinning the flask.After this, it was digested in digestion flask on the heater till the solution may became clear.The process continued for 30 minutes.When the digestion completed, the heater was turned off in order to cool the solution.The digest was then diluted with 50ml distilled water in a volumetric flask.5 ml of the digest was then taken by using pipette.The solution was then transferred into distillation tube.Then, 5 ml of 40 % Sodium hydroxide solution was added through the funnel.Distillate was distilling for five minutes.20ml of 4% boric acid solution was taken in a flask and some drops of indicator were added into the flak and then, collected the distillate.During distillation, the color changed from pink to blue.After that, the distillate was titrated against 0.1 N HCl taken in a burette till the pinkish color restored.The reading of the volume of HCl was noted and % protein was calculated. 21

Procedure
For the extraction of crude fat, the Soxhlet apparatus was used.Here in this process, first 2 gm moisture free sample powder was taken in a clean thimble.The thimble was than plugged with cotton then placed it in extractive tube.Weighted the 250-ml round bottom flask then added petroleum ether solvent up to 1/3 rd of the flask after that fixed it in the extractive tube.Turned on burner and tap water to flow.Run the apparatus for 3 to 4 hours.After that, thimble was removed from the tube; the flask was then placed on the water bath to heat up and to remove the moisture.The flask was then placed in oven at 105C o in order to dry for 1hr.It was allowed to cool and weighted again (W2).

Carbohydrates determination:
Carbohydrates can be determined by adding the weights of all the other contents then subtracted from 100. 24 carbohydrates= (crude fibre+ crude fat+ crude protein+ moisture contents+ Ash) -100

Analgesic activity by writhing induced method Requirements
Acetic acid, plant extract and silver nanoparticles, diclofenac sodium, Albino mice (about 22g b.w), normal saline solution and 3 and 5 cc syringes

Procedure
The animals were not fed anything for at least 24 hours before the start of the experiment.There was a total of six groups.Group I was treated with diclofenic sodium (10mg/kg b.w, p.o) used as positive control.Group II was treated with normal saline (10mg/kg b.w, i.p) used as negative control.Group III was treated with 100mg/kg b.w, i.p of plant extract.
Group IV was treated with 200mg/kg b.w, i.p of plant extract.Group V was treated with 300mg/kg b.w, i.p route of plant extract.Group VI was treated with only acetic acid.After 30 minutes of drug administration such as diclofenac sodium, normal saline solution, plant extract solution to the mice then injected 1 % acetic acid into the peritoneal cavity in order to cause pain.Recorded the writhing (extension of hind limbs, contraction of abdomen and turning of trunk) in the mice after 5, 10 and 15 minutes in various groups.] Pharmacognostic study of Euphorbia dracunculoides L. was carried out to standardized the quality and quantity of the drug.

RESULTS
Pharmacognostic studies of Euphorbia dracunculoides L.
Whole plant extract of Euphorbia dracunculoides L. were evaluated for pharmacognostic study i-e Macroscopic, powder drug study, phytochemical analysis, nutritional and elemental analysis.

Macroscopy
The macroscopic character of the plant Euphorbia dracunculoides L. were observed for odor, shape, taste, colour and texture (Table 1, 2, 3).It helped in the correct identification of the plant.It is an annual herb.Its stem is light green at the top and yellow green at the bottom and 15-30 cm tall, it is much branched from the base.Its leaves are alternate, sessile, simple, dark green in color, its roots are yellowish in color, conical shape, smooth with rootlets.

Microscopic study Powder drug study
Powder drug study of whole plant powder of Euphorbia dracunculoides L. was carried out (Figure 1).It shows different structures in powder form

Macroscopy
That reduces the wrong recommendations of medicinal plants and traditional medicines to high extent, stepwise pharmacognostic investigations, including morphology, anatomy, quantitative microscopy determination such as stomata number, index, vein islet r, veinlet termination number, palisade ratio and qualitative and quantitative phytochemical screening give standardization of the crude drugs.Correct identification, authentication and quality assurance of the preliminary resources as an important requirement to make sure the reproducible quality of phytomedicine which will show the safety and effectiveness of herbal products.
Similar study was also conducted by other researchers.Mughal et al. 27 studied the phytochemical analysis and pharmacognostic study of, Euphorbia hirta, Euphorbia prostrata, Jatropha integerrima, Ricinus communis and Euphorbia splendens (Family: Euphorbiaceae).Sharma and Pracheta, 28 carried out the macroscopic and powder drug study of the leaves of the Euphorbia neriiffolia.They observed that the fresh leaves are simple, with leathery texture, dark green in colour, sub-acute apex, acute base, cuneate shaped, entire margins, glabrous surface, reticulate venation.Kumar et al. 29 evaluated pharmacognostically Euphorbia Hirta plant.The leaves of E. hirta are opposite, oblong or oblong-lanceolate, elliptical, its upper surface is darker, with a faintly toothed margin.Its flowers are numerous, small and about 1 cm in diameter.Kanthale and Panchal, 30 evaluated the pharmacognostic study of Adhatoda vasica Nees.

Microscopic study Powder drug study
Powder microscopy help in the identification of the herbal drugs and detection of adulteration in crude drugs Similar study was also carried by other researchers; Sharma and Pracheta, 28 carried out the macroscopic and powder drug study of the leaves of the Euphorbia neriiffolia.Powder drug study revealed the presence of anomocytic stomata, spongy parenchyma, unicerrate multicellular trichomes, xylum parenchyma, starch grains, xylum fibres, epidermal cells.Patil and Jadhav, 31 carried out the macroscopic and microscopic study of the plant Antidesma acidum Retz.Its powder drug study of leaves showed unicellular trichomes, epidermal cells, phloem fibres, hexagonal sclerenchyma cells, xylem and stomata.Kumar et al. 29 investigated trichomes, vascular bundles, xylem cells, spongy parenchyma Phytochemical Analysis.
Both qualitative and quantitative phytochemical analysis of the ethanolic extract of whole plant Euphorbia dracunculoides L. were carried out.Qualitative analysis of Euphorbia dracunculoides L. revealed the presence of carbohydrates, proteins, flavonoids, phenolic compounds, tannins, alkaloids, glycosides, sterols etc (Table 4).Quantitative analysis of some of the secondary metabolites such as saponins, flavonoids, tannins and alkaloids were also carried out in the ethanolic whole plant extract of Euphorbia dracunculoides L. The quantitative result revealed the presence of sterols (20.5%), tanins (18.5%), saponins (17%) and alkaloids (10%).(Table .5)

Elemental analysis
In the present study, the elemental analysis of Euphorbia dracunculoides L. conducted which showed the presence of both macro and micro nutrients given in the table.7.

Analgesic activity by writhing induced method
Analgesia is the sensation of "pain".Usually a deeply unlikable sensation brought about by internal or external stimulation.An analgesic is used to relieve pain without effecting consciousness or eliminating complete sensation.They act on the CNS or the peripheral nervous system.Analgesic activity is the capability of a substance to ease pain.Acetic acid permeates the capillaries which releases endogenous constituents to give rise inflammatory pain by stimulating nerve endings.Similar study was shown by other researchers; Chandrashekar et al. 48investigated the analgesic activity of the Phyllanthus lawii.They suggested that the methanolic extract of P. lawii reduces the writhing in mice by injecting acetic acid showing peripheral analgesic activity.It releases the pain so it showed excellent analgesic activity.Kadam and Bodhankar, 49 observed the significant analgesic activity at the dose 400mg/kg b.w of the ethanolic and phloem cells in the leaves of Holoptelea integrifolia.Sundar and Pillai, 32 evaluated the pharmacognostical standards by studying macroscopic and microscopic characters of Solanum virginianum L.

Phytochemical Analysis
Phytochemicals are actually organic compounds which possess medicinal properties (Mir et al. 33 Medicinal plants contain some bioactive organic compounds i-e carbohydrates, steroids, tannins, flavonoids, alkaloids and terpenoids, which provide specific physiological action on the human body.Okeniyi et al. 34 38 carried out the qualitative and quantitative analysis of Tanacetumparthenium L. Among quantitative analysis phenols were found maximum in methanolic extract (52.01 mg/g) followed by terpenoids (53.65 mg/g), flavonoids (19.64mg/g) and tannin (16.53 mg/g).Jaradat et al. 39 studied of saponin = W2 -W1 W1 = Weight of filter paper W2 = Weight of filter paper + residue of tannins = W2 -W1 W1 = Weight of filter paper W2 = Weight of filter paper+ residue
of acid 0.014 dilution 100 % protein Wt. of sample aliquot of digest taken for distillation Where S = Sample titration reading B = Blank titration reading 0.14 = Millequivalent of nitrogen Determination of fat or ether extract: Apparatus: Water bath, petroleum ether (B.P 40-60C o ), Soxhlet extraction apparatus, Extraction Thimble (Whatman).

23 %
Fats percentage was then calculated by the following equation 22 % fat (Ether extract) = x Weight of sample × 100 Where X = Weight of the fats = W2 -W1 W1 = Weight of the empty flask W2 = Weight of the empty flask + sample after evaporation of solvent Crude fibre determination: Reagents: Asbestos, Ethyl Alcohol, Petroleum ether, Sodium hydroxide (2%), HCl (2%) Apparatus Muffle furnace, crude fibre extraction apparatus, suction pump, oven, electric balance, Gooch crucible, funnel, beaker, filter paper or cotton clothProcedure 2 gram of sample powder was taken in a beaker and added 200 ml of 2% NaOH kept on water bath for 30 minutes till it boils then filtered it with a cloth.The residue was then transferred into the beaker and add 200 ml of 2% HCl keep on water bath and boil for 30 minutes.Filter it with a cloth again and the remaining residue was then washed with 200ml hot water in order to remove the acid.The residue was then transferred into the crucible and placed in oven at 105C O for 4 hours.After that it was transferred to dessicator for 30 minutes to cool and weighed (W1).Then the crucible placed in a muffle furnace for 4 hours at 550 C O burned till white and weighted again (W2).Crude fibre was then calculated. of sample after placing in oven W2 = Weight of sample after placing in muffle furnace the authentication and standardization of crude plant extract drug.Following were some of the structures observed under microscope.• Fragments of epidermal cells • Stomata • Xylum vessels • Phloem fibres

Figure 1 :
Figure 1: Powder drug study of Euphorbia dracunculoides L. A. Epidermal cells.B. Stomata.C. Multicellular Trichomes.D. E. Phloem Fibers, F. Cortical cells, G. Xylem vessel H. Oil droplets, I. Pollen grain, J. Epidermal cell with stomata, K. Single trichome, L. Starch grains •The test samples were treated with NaOH solution.A strong yellow color is observed.If dilute acid is added, it decolorizes which proves Flavonoid presence.
•If a yellow color is observed once lead acetate solution is added to the test samples, it means flavonoids are present.

Table 3 : Macroscopic studies of Euphorbia dracunculoides Leaf S. No Characteristic Observation
Analgesic activity by writhing induced methodCrude ethanolic extract of Euphorbia dracunculoides at various doses through i.p. route (100, 200, 300mg/kg b.w) showed decrease in the mean number of writhing in different test groups at different time as shown in the table 8.After five minutes, In saline treated group mean writhing was 31.66±1.66.

Table 5 : Quantitative analysis of Euphorbia dracunculoides L
47hedra alata.Their result showed the total phenolic and flavonoid contents present in the extract of Ephedra alata.Food is the basic need of all living organisms.The basic purpose behind eating is getting calories, maintaining, growing, reproducing and multiplying of cells.A food must have proteins, carbohydrates, lipids, mineral elements, vitamins and water.Proteins, fats and carbohydrates are the most important nutrients of life.Similar studies were conducted by other researchers; Offor et al.40analyzed crude fibre (17.3%),Ash (3.8%), moisture (14.8%), carbohydrates (50.6%), fat (4.5%) and protein (24.5%) in the dried leaves of moringa.Igwenyi et al.41conducted the Proximate analysis of Euphorbia hyssopifolia which showed the quantity of crude fat (3.20%), protein (0.88%), ash content (3.80%), carbohydrate content (6.98%), crude fiber (2.15%) and high moisture content (83.00%).Ghani et al.42analyzed 4 medicinal plants i-e Achryanthus aspera, Peganum hermala, Mentha longifolia and Solanum nigrum for nutritional study.Offor and Uchenwoke,43examined the proximate analysis of Gongronema latifolium leaves.Medicinal plants contain Macro and Micro elements that control biochemical processes in the human body.The active ingredients of these plants play an essential role in metabolism.Similar work was also performed by other researchers Saxena et al.44determined the elements in the roots, leaves and stem of Uraria picta.Henry et al.45demonstrated the elemental analysis of Cayratia gracilis (Guill.&Perr.).Barua et al.46studied the elemental analysis of 5 plants i-e Bryophyllum pinnatum, Corchorous fascicularis, Ocimum sanctum, Acorous calamus and Plantago erosa.Parvez et al.47analyzed the macro minerals i-e Ca, N, P, S, Mg and K and micro minerals i-e Zn, Fe, Cl, Mn, Cu, Mo, Se and Na in the Euphorbia granulate.
36nfirmed the presence of reducing sugars, cardiac glycosides, flavonoids, saponins, triterpenes, tannins, steroids, glycosides, carbohydrates and phlobatanins in the stem and leaf extract of Euphorbia heterophylla.Qaisar et al.35studied secondary metabolites of the aerial parts of Euphorbia helioscopia, E. hirta, E. milli and E. prostrate.They showed that these plants contain flavonoids, tannins and terpenoids.Similar study has been carried by other researchers; Pranoothi et al.36carried out the Quantitative analysis of Leucas indica which revealed the presence of total flavonoids and phenols contents in the plant extract.

Table 7 : Elemental Analysis of Euphorbia dracunculoides L.
50tada phaseoloides by performing acetic acid induced pain method.Ramamurthy and Sathiyadevi,50observed the analgesic effect by acetic acid induced writhing method in mice of the plant Indigofera trita.They showed that at the dose 200mg/kg and 400mg/kg b.w the plant extract are more potent to reduce writhingCONCLUSION AND RECOMMENDATIONIt is concluded from the present research work that Euphorbia dracunculoides is an erect glabrous annual or sometimes perennating herb.The powder microscopy revealed presence of various type of tissues that is a tool for the determination of adulterants in crude powder of E. dracunculoides available in herbal market.Qualitative analysis revealed the presence of carbohydrates, proteins, flavonoids, phenolic compounds, tannins, alkaloids, glycosides, sterols and saponins etc.The quantitative result of Euphorbia dracunculoides L. revealed the presence of highest amount of sterols (20.5%).The result of elemental analysis showed highest concentration of macronutrients i-e Magnesium while the highest concentration of micronutrients i-e Iron (3.043±0.0168).Analgesic activity of Euphorbia dracunculoides L. by writhing induced method showed excellent analgesic effect at 300 µg/ml.Hence it is recommended that further advance and high level work should be done on E. dracunculoides to use it as a natural, economic and safe drug instead of synthetic haphazard medicines.