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Studies on phytochemical, antioxidant, anti-inflammatory and analgesic activities of Euphorbia dracunculoides
© Majid et al. 2015
Received: 9 June 2015
Accepted: 21 September 2015
Published: 7 October 2015
Plants provide an alternative source to manage various human disorders due to diverse metabolites. Euphorbia dracunculoides of family Euphorbiaceae is used by local practitioners in rheumatism, epilepsy, edema, snake bite, warts and also possesses diuretic and purgative effects. The present study evaluated the antioxidant, anti-inflammatory and analgesic activities of various extracts of E. dracunculoides. Further, phytochemical constituents of the leading extracts were also investigated.
Dry powder of E. dracunculoides was extracted with n-hexane (EDH), acetone (EDA), ethanol (EDE), ethanol + water (1:1) (EDEW) and methanol (EDM) and screened for phytochemical classes, total phenolic (TPC) and flavonoid content (TFC). Antioxidant effects of the extracts were manifested by in vitro multidimensional assays. The anti-inflammatory and analgesic activities of the extracts were evaluated through carrageenan induced paw edema and hot plate test in rat. In addition, GC-MS analysis of EDH and HPLC-DAD analysis of EDEW was carried out to determine the presence of active constituents.
Qualitative analysis of various extracts of E. dracunculoides assured the existence of tannins and coumarins while presence of anthraquinones and anthocyanins was not traced in these extracts. Maximum quantity of TPC and TFC was recorded in EDEW followed by EDE. EDEW and EDE showed significant antioxidant activities with therapeutic potential against hydroxyl and phosphomolybdate radicals, β-carotene bleaching assay and in reducing of iron while moderate to low scavenging abilities were recorded for DPPH, nitric oxide and for iron chelation. During anti-inflammatory activity after 4 h of drug administration the 300 mg/kg body weight dose of EDH (68.660 ± 10.502 %) and EDE (51.384 ± 8.623 %) exhibited strong anti-inflammatory activity and reduced the carrageenan-induced paw edema in rat as compared to standard drug diclofenac sodium (78.823 ± 6.395 %). Treatment of rats with EDH (70.206 ± 5.445 %) and EDE (56.508 ± 6.363 %) after 90 min showed significant increase in percent latency time in hot plate test as compared to morphine (63.632 ± 5.449 %) treatment in rat. GC-MS analysis of EDH indicated the presence of 30 compounds predominantly of steroids and terpenoids. HPLC-DAD analysis against known standards established the presence of rutin, catechin, caffeic acid and myricetin in EDEW.
Our results suggest that presence of various polyphenolics, terpenoids and steroids render E. dracunculoides with therapeutic potential for oxidative stress and inflammation related disorders.
KeywordsEuphorbia dracunculoides Antioxidant Anti-inflammatory Analgesic Terpenoids Steroids
Medicinal plants play an appealing role in modulation of several human disorders. Many civilizations and cultures like Chinese, Ayurvedic, Unani and Hindi has a confirm belief on these cures to treat their health related issues. Plants derived chemicals are important source of clinical agents that have sedative, anti-depressant, antioxidant, antispasmodic, anxiolytic, anti-inflammatory, immunomodulatory, analgesic, anti-pyretic, and cardio protective activities . This customary herbal medicinal system is deep rooted in their cultures and habitats and knowledge of home remedies is conveyed accordingly to their descendants as the time goes .
During the normal metabolic process human body produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) as byproduct to accomplish the normal physiological processes. Oxidative stress can be declared as a root cause for many diseases especially chronic inflammatory disorders such as rheumatism, diabetes, carcinomas, mutagenesis, sarcomas, aging and circulatory disorders . Living cells possess an excellent defense mechanism to cope with damaging effects of free radicals and this is made possible by a variety of detoxifying enzymes and metabolites inside the body that scavenge free radicals in a professional way. Among them superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) are the main stream enzymes supported with scavengers and chelating agents. The indirectly acting antioxidants are very important in protecting body from deleterious effects of ROS/RNS. This group comprised thousands of compounds involved in free radical scavenging and most of them are derived from dietary sources such as vegetables and fruits.
We are much more endangered by free radicals than being secured by natural scavenging entities producing in our body in stressed conditions. Phytochemicals play a unique role in avoiding hazardous effects of free radicals . Polyphenols are excellent free radical scavengers and inhibitors of lipid peroxidation. Terpenoids and steroids are useful metabolites for various metabolic disorders. These properties make their role crucial from therapeutic and pharmacologic point of views.
The trend of finding the pharmacological activities of the plants of known medicinal uses in folk medicines is quite much effective than going randomly after it [5, 6]. The genus Euphorbia contributes as the largest amongst the spurge family with over 2000 species, with awesome use value in folk Chinese medicinal system used mainly for skin diseases and edemas .
Several species of Euphorbia have been used in local system of medicine; for the treatment of various ailments. Rhizome of Euphorbia neriifolia and aerial parts of Euphorbia royleana has been used for the treatment of anti-inflammatory disorders [8, 9]. In local system of medicine such as Africa and Australia, Euphorbia hirta has used as a remedy for various ailments especially in hypertension and edema. Previous studies have evaluated E. hirta for antipyretic, analgesic, anti-inflammatory and diuretic activities [10, 11]. Strong antioxidant activity of E. macroclada and E. acanthothamnos has been determined in previous studies .
Euphorbia dracunculoides Lam., (Euphorbiaceae) is distributed in Southwest Asia, North Africa and South Europe. It is an annual herb and is usually found along riverbanks, in valleys and roadsides of sandy areas in Khyber Pakhtunkhwa Province of Pakistan. Fruits are used to remove warts from skin . Leaves are used in snake bite and epilepsy . A decoction of whole plant is applied on body of cattle for lice killing . Euphorbia dracunculoides has been used by local practitioners for its diuretic and purgative properties. Structurally diversified 19 diterpenoids have been isolated from aerial parts of E. dracunculoides . Because of similar morphology of the dried aerial parts of E. drancunculoides to Ruta graveolens, it is sold or used clinically as replacement of R. graveolens for analgesic and inflammatory disorders; gout and arthritis [17, 18]. To our knowledge the scientific validation of E. dracunculoides for the use in inflammation related disorders has not been reported earlier. For this purpose we investigated preliminary phytochemical composition, antioxidant and anti-inflammatory activities of various extracts of E. dracunculoides. EDEW showed significant antioxidant activity; was subjected to HPLC-DAD analysis for the presence of flavonoid constituents. EDH exhibited remarkable analgesic and anti-inflammatory activities; was explored for GC-MS analysis.
Preparation of extract
Plant was recognized by its local name and collected from the District Lakki Marwat in March 2014. The plant sample after authentication was deposited (Accession No. 127962) at the Herbarium of Pakistan, Quaid-i-Azam University, Islamabad, Pakistan. The fully shade dried aerial parts of E. dracunculoides were first powdered followed by two extraction (36 h) with n-hexane, acetone, ethanol, ethanol + water (1:1 v/v) and 95 % methanol in 2 : 1 ratio (v/w). Filtered extracts; EDH, EDA, EDE, EDEW and EDM were dried under vacuum in a rotary evaporator at 40 °C and stored at 4 °C for in vitro and in vivo experiments.
Different qualitative tests were employed to identify the phytochemical classes present in various extract of the plant E. dracunculoides.
Assessment of phenols
For the presence of phenols previously reported methodology was followed . Each sample (1 mg) was suspended in 2 ml of distilled water containing 10 % ferric chloride. The confirmation sign for the presence of phenol was the development of blue or green color.
Assessment of flavonoids
Protocol of Trease and Evans  was followed to establish the presence of flavonoids in each sample. Briefly, 1 mg of each sample was allowed to react with 1 ml of 2 N sodium hydroxide and appearance of yellow color was considered as the confirmation sign of flavonoid presence.
Assessment of coumarins
An aliquot of each sample (1 mg/ml) was mixed with 1 ml of 10 % sodium hydroxide. Appearance of yellow color formation in the test tube was the proof of coumarins presence in test sample .
Assessment of saponins
Each sample (2 mg) was suspended in 2 ml of distilled water and vigorously shaken. The formation of a soapy layer of almost 1–2 cm was the indication of saponins presence .
Assessment of tannins
Confirmative sign of tannins was the development of dark blue or greenish black color on the mixing of 1 mg of each sample and 2 ml of 5 % ferric chloride .
Assessment of terpenoids
Each sample 0.5 mg was mixed with 2 ml of chloroform and 2 ml of concentrated sulphuric acid. The appearance of a red brown colored layer in the middle of two layers confirmed the existence of terpenoids .
Assessment of anthraquinone
Development of red color was considered as indication for the presence of anthraquinone after mixing of 1 mg of each sample with 2 ml of diluted 2 % hydrochloric acid .
Assessment of anthocyanin and betacyanin
Each sample (1 mg) was boiled for 10 min in 2 ml of 1 N sodium hydroxide. Formation of bluish green color was the sign of anthocyanin and yellow color formation of betacyanin presence .
Assessment of alkaloids
An amount of 2 mg of each sample was mixed with concentrated sulphuric acid. The reaction mixture was allowed to react with Mayer’s reagent. Appearance of green color or formation of white precipitates was the symbol of alkaloid presence .
Total phenolic as well as flavonoid contents were quantified by the following narrated procedures.
Total phenolic contents (TPC)
To determine the total phenolic content in each sample spectrophotometric method already reported was followed . Briefly, 1 ml of each sample (1 mg/ml) was mixed with 9 ml of distilled water and I ml of Folin-Ciocalteu reagent. The mixture obtained was mixed rigorously for 5 min and 10 ml of 7 % Na2CO3 was added to the mixture. By the addition of distilled water the final volume of mixture was made to 25 ml and was incubated at room temperature for 90 min. Optical density was ensured at wavelength of 750 nm in triplicate for each sample. By using gallic acid as standard the estimation of TPC was carried out as mg of gallic acid equivalents (GAE) per gram of dry extract/fraction.
Total flavonoid content (TFC)
To estimate the TFC in the test samples, 0.3 ml of each sample was mixed with 0.15 ml of 0.5 M NaNO2 followed by the addition of 0.1 ml of 0.3 M AlCl3.6H2O, and 3.4 ml of 30 % methanol . An aliquot of 1 ml of 1 M NaOH was added to it after a lapse of 5 min. The optical density of the reaction mixture was recorded at 506 nm wavelength against the reagent blank. Total flavonoid content as mg rutin equivalents per gram of dry extract/fraction was estimated while using the calibration curve of rutin.
Gas chromatography-Mass spectrometry (GC-MS) analysis
EDH was analyzed for the presence of active constituents on “Thermo GC-Trace Ultra Ver; 5.0” gas chromatograph coupled with a “Thermo MS DSQ II” for mass determination. Components were separated on a “ZB 5-MS Capillary Standard Non-polar Column” with 60 m length having 0.25 μm film thickness. During the experiment the temperature was raised from 70 to 260 °C at a rate of 6 °C/min. The flow rate of the carrier gas; helium was 1 ml/min while injection volume of the samples was 1 μl. The identification of chemical constituents was based on comparison of their relative retention times and mass spectra with those obtained from authentic sample and/or the NIST/NBS and Wiley libraries spectra .
High performance liquid chromatography (HPLC) analysis
On account of significant antioxidant activity for most of the in vitro assays the EDEW extract was selected for HPLC-DAD analysis. HPLC analysis of EDEW was carried out by using HPLC-DAD (Agilent Germany) equipment using Sorbex RXC8 (Agilent USA) analytical column with 5 μm particle size and 25 ml capacity. Mobile phase was consisted of eluent A, (acetonitrile-methanol-water-acetic acid /5: 10: 85: 1) and eluent B (acetonitrile-methanol-acetic acid/40: 60: 1). The gradient (A: B) utilized was the following: 0–20 min (0 to 50 % B), 20–25 min (50 to 100 % B), and then isocratic 100 % B (25–40 min) at flow rate of 1 ml/min. The injection volume of the sample was 20 μl. Before the injection samples were filtered through 0.45 μm membrane filter. Among the standards rutin and gallic acid were analyzed at 257 nm, catechin at 279 nm, caffeic acid at 325 nm and quercetin, myricetin, kampferol were analyzed at 368 nm . Each time the column was reconditioned for 10 min before the next analysis. All samples were assayed in triplicates. Quantification was carried out by the integration of the peak using the external standard method. All chromatographic operations were carried out at an ambient temperature.
In vitro antioxidant assays
The in vitro antioxidant assays were carried out by preparing the plant samples (1 mg/ml) in 95 % methanol and then making its serial dilutions. The specific protocol was followed for finding specific scavenging activities of the plant samples.
DPPH (1, 1-diphenyl-2-picryl-hydrazyl) radical scavenging assay
Hydroxyl radical scavenging assay
The power of scavenging hydroxyl free radicals refers to the antioxidant potential of plant samples using methodology practiced by . This technique involved the mixing of 500 μl of 2-deoxyribose (2.8 mM) prepared in 50 mM phosphate buffer and its pH was maintained at 7.4. The reaction mixture was prepared by addition of 100 μl of 0.1 M EDTA, 200 μl of ferric chloride (100 mM) and 100 μl of 200 mM H2O2 and 100 μl of plant sample.
Nitric oxide scavenging assay
Chelating power assay
β-Carotene bleaching assay
Where AA (120) is the absorbance of the antioxidant at t = 120 min, AC (120) is the absorbance of the control at t = 120 min, and AC (0) is the absorbance of the control at t = 0 min.
Reducing power assay
Reducing power of various extracts was estimated by the method of [30, 31]. Briefly, 2 ml of plant extract was mixed with 2 ml of 0.2 M phosphate buffer (pH 6.6) and 2 ml of potassium ferricyanide (10 mg/l) and the reaction mixture was incubated at 50 °C for 20 min. After addition of 2 ml of trichloroacetic acid (100 mg/l) in the reaction mixture, 2 ml was of it was diluted with 2 ml of distilled water and 0.4 ml of FeCl3 (0.1 %). Optical density of the reaction mixture was measured at 700 nm after 10 min of incubation. Gallic acid was used as standard.
The methodology of  was used to assess the antioxidant capabilities of the plant sample. Accordingly, 0.1 ml of the plant sample was mixed with 1 ml of the reagent solution (prepared by adding 28 mM Na3PO4 and 0.6 M H2SO4 with that of 4 mM ammonium molybdate). After incubation at 95 °C in a water bath for 90 min the reaction mixture was cooled down to room temperature and optical density was recorded at 765 nm. Ascorbic acid served as standard in this assay.
EVc = Edema volume of control animals, EVt = Edema volume of test sample animals.
Data obtained in this study was presented as mean ± SD. One way analysis of variance was performed to determine the variability among groups by Statistix 8.1. GraphPad Prim 5 was used to determine the correlation of IC50 values of antioxidant assays with TPC and TFC by Pearson’s correlation coefficient. Significant differences among groups were calculated by Tukey’s multiple comparison tests. Statistical significance was set at P > 0.05.
Extraction yield, total phenolic and flavonoid content
Extraction yield, total phenolic and flavonoid contents of E. dracunculoides
Yield (mg/100 g powder)
Total phenolic content (mg gallic acid equivalent/ g dry sample)
Total flavonoid content (mg rutin equivalent/ g dry sample)
8.21 ± 0.49e
4.18 ± 0.25e
10.62 ± 0.33d
4.52 ± 0.37d
16.41 ± 0.54b
6.77 ± 0.31b
17.35 ± 0.62a
7.57 ± 0.42a
14.11 ± 0.37c
4.84 ± 0.29c
Phytochemical analysis of E. dracunculoides
GC-MS analysis of n-hexane extract
GC-MS analysis of EDH
2,4-Decadienal, (E,E)- (Cas)
Phenol, 2-Methoxy-4-(2-Propenyl)- (Cas)
Hexadecanoic acid, methyl ester (CAS)
Hexadecanoic acid (CAS)
Hexadecanoic acid, ethyl ester
9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-(CAS)
1H-Imidazole, 1-ethyl- (CAS)
2-Acetamido-3-(3,4-dihydroxyphenyl) propenoic acid
2,7-Diphenyl-1,6-dioxopyridazino[4,5-2′,3′] pyrrolo[4′,5′-D pyridazine
Stigmasta-5,22-dien-3-ol, (3á,22E)- (CAS)
HPLC-DAD analysis of ethanol + water extract
HPLC-DAD results for EDEW of E. dracunculoides
Quantity (μg/mg dry extract)
65.8 ± 2.2
15.3 ± 1.2
8.5 ± 0.75
4.54 ± 0.35
In vitro antioxidant activities
DPPH radical scavenging activity
IC50 values of different antioxidant activities of E. dracunculoides extracts
297.12 ± 3.22a
756.31 ± 4.23a
244.06 ± 3.81a
616.57 ± 3.33a
202.20 ± 3.41b
583.40 ± 3.65b
181.10 ± 1.93c
531.43 ± 2.94b
263.61 ± 3.71b
121.70 ± 2.05c
366.81 ± 3.12d
120.53 ± 2.52d
279.51 ± 2.57e
144.71 ± 3.44c
78.02 ± 1.21d
317.80 ± 3.35e
100.40 ± 1.81e
331.46 ± 2.42d
351.33 ± 4.54a
142.43 ± 2.51c
405.01 ± 3.57c
210.23 ± 3.41b
446.3 ± 3.52c
80.74 ± 2.25d
26.65 ± 2.41d
244.45 ± 2.35f
45.97 ± 1.92e
189.85 ± 1.53f
78.57 ± 2.15f
Correlation of IC50 values of different antioxidant activities of E. dracunculoides with total phenolic and total flavonoid contents
DPPH radical scavenging activity
Hydroxyl radical scavenging activity
Iron chelating assay
Nitric Oxide radical scavenging Activity
β - carotene bleaching scavenging activity
Reducing power assay
Hydroxyl radical (•OH) scavenging assay
All the extracts of E. dracunculoides scavenged •OH radicals and prevented 2-deoxyribose breakdown (Table 5). A concentration-dependent pattern was observed for hydroxyl radical scavenging activity. Lowest IC50 values were recorded for EDEW (78.02 ± 1.21 μg/ml) followed by EDE (121.70 ± 2.05 μg/ml), EDM (142.43 ± 2.51 μg/ml), EDA (202.20 ± 3.4 μg/ml) and EDA (297.12 ± 3.22 μg/ml). IC50 values of EDH, EDA, EDE, EDEW and EDM were significantly higher from rutin (80.74 ± 2.25 μg/ml) and gallic acid (45.97 ± 1.92 μg/ml). Significant correlation of IC50 values of hydroxyl radical scavenging was determined for TPC (R2 = 0.9505, P < 0.01) as well as for TFC (R2 = 0.7224, P <0.05) (Table 6).
Nitric oxide (NO−) scavenging assay
In the present study, moderate level of nitric oxide scavenging activity was observed for all the extracts with IC50 values for EDEW (317.8 ± 3.35 μg/ml), EDE (366.81 ± 3.12 μg/ml) and EDM (405.01 ± 3.57 μg/ml) as compared to standard ascorbic acid (244.45 ± 2.35 μg/ml). IC50 values for other extracts were 583.4 ± 3.65 μg/ml and 756.31 ± 4.23 μg/ml for EDA and EDH, respectively (Table 5). IC50 values obtained for nitric oxide scavenging activity exhibited a significant correlation with TPC (R2 = 0.9638, P <0.01) and TFC (R2 = 0.6822, P <0.05) (Table 6).
Iron chelating activity
Iron chelating activity (IC50 values) of E. dracunculoides extracts are given in Table 5. In current study, the best IC50 values for iron chelation were exhibited by EDE (279.51 ± 2.57 μg/ml) while the least by EDH (616.57 ± 3.3 μg/ml). IC50 value for other extracts was; EDEW (331.46 ± 2.42 μg/ml), EDM (446.3 ± 3.52 μg/ml) and EDA (531.43 ± 2.94 μg/ml). IC50 of standard EDTA was 189.85 ± 1.53 μg/ml (Table 5). Significant correlation (R2 = 0.9361, P < 0.01) of IC50 values was observed with TFC (R2 = 0.9361, P < 0.01) and for TPC (R2 = 0.8232, P < 0.05) (Table 6 ).
β-Carotene scavenging activity
Ethanol + water extract of E. dracunculoides (EDEW) showed the lowest IC50 value (100.40 ± 1.8 μg/ml) as compared to other extracts viz. EDE (120.53 ± 2.52 μg/ml), EDM (210.23 ± 3.41 μg/ml) and EDA (181.10 ± 1.9 μg/ml). However, the maximum IC50 was shown by EDH (244.06 ± 3.8 μg/ml) as compared to the standard catechin (78.57 ± 2.15 μg/ml) shown in Table 5. The concentration dependent inhibition in β-carotene bleaching power pattern was observed for all the extracts. The assay showed significant correlation of IC50 with both TPC (R2 = 0.7579, P < 0.05) and TFC (R2 = 0.8965, P < 0.01) as shown in Table 6.
Reducing power assay
Total antioxidant capacity (phosphomolybdenum assay)
Anti-inflammatory activity of E. dracunculoides
Effect of E. dracunculoides on carrageenan-induced paw edema in rat
Edema volume (ml)/ Percent edema inhibition
DMSO (2 ml)
0.506 ± 0.046
0.776 ± 0.045
0.710 ± 0.021
0.671 ± 0.017
0.600 ± 0.021
0.551 ± 0.056
0.706 ± 0.056
0.640 ± 0.049
0.613 ± 0.049
0.586 ± 0.056
42.862 ± 5.614ab
56.903 ± 8.443ab
69.975 ± 5.730a
78.823 ± 6.395a
0.548 ± 0.022
0.685 ± 0.033
0.630 ± 0.021
0.615 ± 0.018
0.600 ± 0.023
49.652 ± 7.649a
60.171 ± 3.690a
67.524 ± 8.004a
68.660 ± 10.502a
0.548 ± 0.035
0.751 ± 0.027
0.696 ± 0.030
0.683 ± 0.032
0.651 ± 0.031
24.960 ± 7.283c
27.492 ± 5.730d
34.028 ± 9.170c
37.156 ± 6.297c
0.560 ± 0.037
0.730 ± 0.016
0.676 ± 0.037
0.651 ± 0.039
0.640 ± 0.026
37.307 ± 8.113abc
43.014 ± 12.271bc
55.269 ± 7.215ab
51.384 ± 8.623b
0.520 ± 0.046
0.705 ± 0.045
0.643 ± 0.030
0.633 ± 0.044
0.608 ± 0.039
31.752 ± 3.883bc
39.746 ± 10.125cd
44.648 ± 9.127bc
46.302 ± 8.975bc
0.545 ± 0.032
0.738 ± 0.026
0.683 ± 0.031
0.676 ± 0.026
0.653 ± 0.039
28.665 ± 11.396c
32.394 ± 8.443cd
35.662 ± 12.938c
34.107 ± 8.975c
Analgesic activity of E. dracunculoides
Analgesic activity of E. dracunculoides of hot plate test in rat
Latency time sec
DMSO (2 ml)
9.000 ± 0.894
9.833 ± 1.169
10.167 ± 0.752
10.167 ± 0.752
3.971 ± 3.588d
5.490 ± 3.378e
5.490 ± 3.378e
9.333 ± 1.032
21.000 ± 1.549
25.667 ± 1.032
22.500 ± 1.048
56.160 ± 9.114b
78.889 ± 5.853a
63.632 ± 5.449ab
9.833 ± 1.169
21.667 ± 1.633
24.833 ± 1.169
24.000 ± 1.095
58.304 ± 9.974a
74.309 ± 5.864b
70.206 ± 5.445a
9.666 ± 0.816
14.167 ± 1.602
15.167 ± 1.169
16.833 ± 0.752
22.164 ± 6.680c
26.964 ± 6.193d
35.223 ± 3.210d
10.000 ± 0.894
16.000 ± 1.414
22.333 ± 1.211
21.333 ± 1.032
29.804 ± 8.470bc
61.811 ± 4.528b
56.508 ± 6.363bc
9.666 ± 0.816
15.500 ± 1.048
19.333 ± 1.633
19.667 ± 1.032
28.676 ± 4.528bc
47.331 ± 9.410c
49.204 ± 4.254c
9.666 ± 0.816
17.833 ± 1.472
20.000 ± 1.788
19.500 ± 1.048
40.196 ± 6.553b
50.959 ± 7.486bc
48.248 ± 5.943c
The role of medicinal plants in curing diseases is increasing due to the presence of versatile compounds that have the ability to cure a variety of diseases and helping physicians to cope with increasing ratio of ailments these days . Plants are rich in variety of compounds with different polarities . Maximum yield was obtained in EDEW followed by EDM and the minimum yield was recorded in EDE following the rule that polar solvents dissolve more compounds in comparison to that of non-polar in short time at room temperature . Present studies are in agreement to the concept of polarity based extraction with the experiment run by  on Maytenus royleanus leaves and  evaluating Rumex hastatus roots.
Preliminary qualitative phytochemical screening gives a clue for the medicinal aptitude of the herb. In the conducted study bioactive components that impart biologically active nature to the plant were screened and results ensured the presence of terpenoids, coumarins, flavonoids, tannins, phenols, alkaloids, saponins, and betacyanin. These variant compounds ranging from low polarity to high polarity were extracted in their respective solvents.
The EDH was analyzed through GC-MS and it was found to contain 30 chemical constituents eluted between 6.36 and 40.10 min. Maximum proportion was contributed by terpenoids followed by lactones, steroids, phenols and heterocycles. GC-MS analyses of EDH validated the concept of polarity based extraction as the most of the compounds identified were of non-polar nature.
In the present study through HPLC analysis of EDEW presence of four compounds; rutin, catechin, caffeic acid and quercetin was evaluated. Rutin was present in maximum amount (65.8 ± 2.2 μg/mg dry extract) followed by catechin (15.3 ± 1.2 μg/mg dry extract). Rutin is a known and well reputed secondary metabolite of plants with admirable hepatoprotective, anti-inflammatory and antioxidant activities . Another well-known polyphenolic compound, catechin has good antioxidant potential and provides a reliable defense wall against free radicals. Catechin provides protection against neurological disorders, inflammation and apoptosis. The admiring amount of standard antioxidant phenolic compounds in EDEW is the justification of raised antioxidant potential of this extract. Significant correlation between TPC and TFC with that of antioxidant assays also validates the HPLC results.
Natural antioxidants have attained worth reputation in treating several diseases and have raised the value of folk herbal medicines in the modern era. The ability of DPPH radical scavenging is considered as a milestone while assessing the antioxidant aptitude of a crude plant extract or an isolated pure compound. It is a very short timed assay for appraising the antioxidant abilities and has worth economic values . In this study EDEW showed the maximum scavenging activity with lowest IC50 value, followed by EDE < EDM < EDA < EDH. Greater quantity of phenolics and flavonoids extracted in EDEW might exhibit good scavenging abilities due to donation of electron or hydrogen to stabilize DPPH free radicals. None of the five extracts showed IC50 below ascorbic acid used as standard. Though  and  used different techniques for extraction yet our results are consolidating their findings. The results reported in this study indicated a good correlation with the TFC while non-significant correlation was recorded with TPC. The strong correlation of IC50 values with TFC might be attributed by the presence of active flavonoids such as myricetin, rutin, caffeic acid and catechin.
Antioxidant potential of a plant crude extract or compound can also be estimated by its ability to oxidize linoleic acid. β-carotene is a fat soluble hydrocarbon with yellowish orange color. Linoleic acid hydroperoxides on reaction with β-carotene bleaches its color. This is due to the fact that β-carotene forms a complex with linoleic acid and oxidizes it. This consumption of β-carotene causes the reduction of bright yellow color to light milky color. Now the presence of an antioxidant in the reaction mixture checks the consumption of β-carotene by acting on linoleic acid free radicals. As the β-carotene is released from the complex between it and linoleic acid, the yellow color of the reaction mixture is regained. Hence stronger the antioxidant present in the reaction mixture brighter will be the color and higher will be its optical density . In the present study, EDEW and EDE showed the best activity with lower IC50 values. Significantly good correlation of IC50 values was observed with TPC (P < 0.01) and TFC (P < 0.05) which defines that more active constituents exhibiting inhibition of β-carotene bleaching belong to the TPC while constituents having moderate activity are present in TFC.
Hydroxyl radical is generated during various biochemical reactions in the body. It is a short lived, very toxic free radical having affinity for biomolecules like lipids, proteins, amino acids, sugars, deoxyribonucleic acids, leading to cancer, mutagenesis and cytotoxicity . Superoxide dismutase converts superoxide radical into hydrogen peroxide which is converted to a highly reactive hydroxyl radical. Evidence of •OH scavenging activity by various extracts was obtained through deoxyribose system . In vivo hydroxyl radicals are probably produced through Haber–Weiss reaction where Fe+3 is reduced to Fe+2 with the help of O2•- which leads to initiation of Fenton reaction between H2O2 and Fe+2 . In the present study EDEW and EDE extracts showed the best activity with lower IC50 values. A significant correlation of IC50 values was present with both TPC and TFC. Our results are in well accordance to the findings of  who reported methanol extract of Dicliptera roxburghiana as the most active to scavenge hydroxyl radicals.
At pH 7.4 a vigorous generation of nitric oxide from sodium nitroprusside occurs which further in favorable conditions viz aerobic conditions and aqueous solution react with oxygen and convert to nitrite ions, that can be appraised by Griess reagent. These nitrite ions impart pink color to the reaction mixture. Entities with NO− scavenging abilities hinder the nitrite ion production by consuming the available oxygen. In our study EDEW and EDE showed comparatively good results than rest of the extracts and significant correlation of IC50 values was observed with TPC and TFC. This is due to the fact that EDEW and EDE are rich in bioactive phenolic and polyphenolics which have a fine tendency to scavenge NO− free radicals that are responsible for oxidative stress. Research of  and  is in agreement to our findings.
Idea of iron chelating assay is based on the principle of scavenger’s ability to decolorize the iron-ferrozine complex. Ferrozine quickly reacts with the iron (II) to form water soluble colored complex. Scavenging entity present in the extract forms chelates with iron (II) hindering the iron-ferrozine complex formation and ultimately lowering the color intensity of the solution. In the present study EDE exhibited the best performance with lowest IC50 among all extracts in comparison to EDTA used as standard. Significant correlation of IC50 values was expressed with TFC (P < 0.01) and with TPC (P < 0.05) indicating that more active flavonoids constituents for iron chelation activity.
The basic principle of phosphomolybdate assay is that antioxidant species reduces Mo (IV) to Mo (V) and this reduced form of Mo forms complex with phosphate at acidic pH and raised temperature impart dark green color to the final solution . The electron/ hydrogen donating pattern of antioxidants depends upon its structure and series of redox reactions occurring in the activity . EDEW and EDE showed admiring results than that of EDM, EDA and EDH. The assay showed a good correlation with TPC as well as TFC. Jan et al.  also reported aqueous extract as the best extract in phosphomolybdenum assay, followed by methanol. Moreover, significant correlation has also been reported with TPC which is in consensus with the present study but the method of extraction was a bit different, which suggests that this methodology made no difference in extracting bioactive compounds responsible for total antioxidant activity of the plant extract.
Reducing power of E. dracunculoides was determined by using the potassium ferricynide reduction method. In this assay iron (Fe+3) in ferric chloride is converted to ferrous (Fe+2) by antioxidant compound/extract resulting in conversion of yellow color of the test solution to green. The intensity of green colour is directly proportional to the reducing power of the sample. Basically reducing power of the sample is due to hydrogen donating ability of antioxidants to the free radicals . The assay was significantly correlated with TPC and TFC. This assay results in a pattern of EDEW > EDE > EDM > EDA > EDH at 250 μg/ml. Our study has been supported by the report of  that crude methanol extract was the best sample in reducing power assay after butanol.
Carrageenan is widely used to induce paw edema in rodents to demonstrate anti-inflammatory effect of drugs or herbs. Carrageenan-induced inflammation is considered to be a biphasic model. Initial stage (1–2 h) contributes to the release of histamine, bradykinin and serotonin which mediates the increased synthesis of prostaglandins from surrounding tissues of the injured site. The later phase (3–4 h) is characterized by the elevated level of prostaglandins mediated by the elevated release of leukotrienes and bradykinin. During this phase the cyclo-oxygenase-2 (COX-2) converts arachidonic acid into prostaglandins which is a key factor of inflammation maintenance. In this experiment carrageenan induced edema in the hind paw of rats was inhibited by all the extracts. EDH significantly inhibited the edema formation; 1, 2, 3 and 4 h after the injection of carrageenan in the hind paw of rats. These results indicated that the phyto-constituents present in EDH inhibited the inflammatory mediators of the initial as well as late phase of inflammation induced with carrageenan. The other extracts however, more effectively inhibited the edema during the initial phase. Steroids are established anti-inflammatory agents which inhibit the production of prostaglandin not only by inducing the biosynthesis of phospholipase A2 inhibitor but also by raising the level of cyclo-oxygenase/PGE isomerase [54–56]. In the present investigation GC-MS analysis of EDH revealed the existence of stigmasta-5, 22-dien-3-ol, (3á,22E)- (CAS); a phytosterol possesses strong anti-inflammatory and analgesic properties probably contributing towards the anti-inflammatory properties of EDH [57, 58]. The standard drug also exhibited the strong anti-inflammatory potential after 2, 3 and 4 h of carrageenan injection to rats. Diclofenac sodium like other NSAIDs targets the COX-2 enzyme thereby inhibiting the formation of the paw edema. The anti-inflammatory results obtained with EDH in both phases might be attributed by the counteraction of anti-inflammatory agents such as sterols and terpenoids . The anti-inflammatory effects obtained by the polar extracts; EDE, EDA, EDEW and EDM during the initial phase might be attributed by the presence of flavonoids (rutin, catechin, caffeic acid and myricetin) and other constituents. Anti-inflammatory activities of rutin, catechin and caffeic acid have been well documented . Our studies are in consensus with  and [61, 62] who generated the same anti-inflammatory results of Acacia hydaspica and Boerhavia procumbens in rats.
Thermal nociception models such as, hot plate test was used to evaluate the central analgesic activity. In this study all the extracts of E. dracunculoides showed analgesic effect in the hot plate test. EDH exhibited the best potent analgesic activity among the extracts with 74.309 ± 5.864 % inhibition of pain sensation followed by EDE (61.811 ± 4.528 %) in comparison to morphine (78.889 ± 5.853 %) after 60 min of drug administration. Morphine induces analgesic effect through activation of opioid receptors and the apparent similarity between the results of extracts with standard morphine, indicates that they might work in a same manner to reduce pain sensation. The presence of steroids in EDH might induce the biosynthesis of phospholipase A2 inhibitor and cyclo-oxygenase/PGE isomerase which in turn inhibits the pain producing prostaglandins. Our results are in agreements to the findings of Mondal et al.  evaluating Alternanthera sessilis as analgesic agent. Backhouse et al.  also reported the same results while evaluating Buddleja globosa as analgesic and anti-inflammatory agent.
The present results suggest that the flavonoids in E. dracunculoides might be the key players in scavenging of oxidative stress inducing species while flavonoids along with sterols and terpenoids alleviate the inflammation and pain inducing mediators.
MRK is strongly acknowledged for his kind supervision, expert guidance and generous facilitation of all necessary materials and equipments. Dr. Bushra Mirza is also highly acknowledged for her assistance in HPLC.
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