PHYTOCHEMICAL ANALYSIS , ANALGESIC , ANTI-INFLAMMATORY AND ANTI BACTERIAL ACTIVITIES OF BERBERIS LYCIUM

٭ Shakir Ullah 1 , Gul Jan 1 , Farzana Gul Jan 1 , Siraj Khan 1 ,Maria khattak 1 , Hamida Bibi 2 and Mohsin ihsan 1 . 1. Abdul Wali Khan University, Department of Botany Garden Campus, Mardan, Pakistan 2. Hazara University Mansehra. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

In the present research work the phytochemical investigation of methanolic, ethanolic and chloroform extracts ofBerberis lycium and pharmacological activities of methanolic extracts (Anti-inflammatory and analgesic activity) and antibacterialactivities in methanolic, ethanolic and chloroform extracts was carried out. The phytochemicals analysis showing the presence ofcarbohydrates, flavonoids, phlobatannins, alkaloids, saponins, tannins, phenols, terpenoids, cardiac glycosides was present in methanolic and ethanolic extracts, while alkaloids, phlobatannins, glycosides and protein were absent and quantative phytochemistry showed the flavonoids in chloroform extract as (14.20±0.15mg/ml), Alkaloids (12.10±0.15mg/ml), phenolics (10.45± 0.10mg/ml), Saponins (06.22±0.14mg/ml) and Tannins (04.60±0.65 mg/ml). The pharmacological activities such as Antiinflammatory, at the doses of 600 mg/kg b.w the administration of extract produced a significant anti-inflammatory activity at 3 hours with paw oedema inhibition of 59 % respectively, while the standard drug aspirin inhibited paw oedema of 68%. In analgesic activity the results showed significant dose dependent % inhibition of pain responses in extract 600mg/Kg is 61%. In antibacterial activity the most active among the extracts was with (17.00±0.48 mm) zone of inhibition at the concentration of18 mg/µl against Pseudomonas aeroginosa. Fallowed by Escherchia coli (16.27±0.93mm), Shigella flexneri (16.20±1.89mm) and Salmonella typhi (16.11± 0.82) with concentration of 12 mg/µl.

Tests for Alkaloids:-
For detection of alkaloids, a few drops of Wagner's reagent (Potassium iodine) are add to 2 ml of all three methanol, ethanol and aqouse extracts. The presence of alkaloids was confirmed by the formation of reddish brown precipitate (Khandewal et al., 2015).

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Tests forTannins:-For the detection of tannins Ferric chloride test was done. Ferric chloride (FeCl 3 ) solution was mixed with all three extracts separately. Formation of blue green coloration indicated the presence of tannins. (Kokate et al., 2008).

Tests for Phlobatannins:-
In test tubes 0.5 ml of all the three extracts was taken separately, added 3ml distilled water and shaken for a few minutes then 1% aqueous hydro chloride (HCl) was added and boiled on water both. The presence of phlobatannins is indicated by the formation of red color (Wadood et al., 2013).

Tests for Flavonoids:-
For flavonoids detection, sodium hydroxide (NaOH) solution was added to all the three extracts of the plant. Red precipitation formation of indicate the presence of flavonoids (Kokate et al., 2008).

Tests for Carbohydrates:-
For detection of carbohydrates, 0.5 ml of all three extracts were treated with 0.5 ml of Benedict's regent.Solution were heated for 2 minutes on a water bath. By the formation of reddish brown precipitate the presence of carbohydrate was confirmed (Bussau et al., 2002).

Tests for Phenols:-
For phenol detection, 2 ml of ferric chloride (FeCl 3 ) solution was added to 2 ml ofall the threeextracts separately in a test tube. Deep bluish green colorationformations indicated the presence of phenol (Dahiru et al., 2006).

Tests for Saponins:-
For the detection of saponin, in test tube 5 ml of all three extracts were shaken vigorously. the presence of saponins was confirmed by froth formation (Rajesh et al.,2016).

Tests for (Cardiac) Glycosides:-
For cardiac glycosides detection, 2 ml of all three extracts solution were shaken with 2 ml of glacial acetic acid than added few drops of concentrated sulphuric acid (H 2 SO4) and iron tri chloride (FeCl 3 ). Brown ring formation indicated the presence of Cardiac glycosides (Soni et al., 2011).

Tests for Proteins:-Xanthoproteic test:
For the detection of protein1 ml from of all three extracts were treated with 1ml of concentrated nitric acid (HNO 3) solution. The presence of proteins indicated by the formation of yellow color (Rajesh et al., 2016).
Tests for Terpenoids:salkowski test:-One ml of plant extracts (methanol, ethanol and chloroform) was added with 2 ml of chloroform and carefully added concentrated sulphuric acid (H 2 SO 4 ) along the sides of tube to form a layer.The reddish brown coloration formation indicated the presence of terpenoids (Dahiru et al., 2006).

Tests for Glycosides:-
For the detection of glycosides, 5% of Ferric chloride solution and 1 ml glacial acetic acid were added to 5 ml of all three extracts and then further addition of few drops of concentered sulphuric acid (H 2 SO 4 ). The presence of glycosides was conformed through the formation of greenish blue color (Rajesh et al., 2016).
Quantitative Analysis:-Determination of total flavonoids contents:-Ethanol, methanol and chloroform extracts were used for the detection of total flavonoids contents. Total flavonoids quantification was done by taking 0.5 g of plant extracts. Than the sample were mixed with 4.3 ml methanol and then more addition of 0.1 ml of aluminum tri chloride from 10% prepared solutions of aluminum tri chloride laterally. Potassium acetate (0.1 ml) was added the volume was reached to 5 ml. The mixtures were shaken by vortex to make uniform solution and then these mixture were placed at room temperature for 30 minutes for the purpose of incubation. After the completion of incubation process, the absorption was checked at 415 nm in spectrum. The Quercetin was used as a standard (Daffodil et al ., 2013).

Determination of Total Phenolic Contents:-
Total phenolic quantification was done by the addition of 0.5g plant extract to 1 ml of 80% ethanol. Then the mixture were centrifuged for 15 minutes at 12,000 rpm. After that the supernatant were kept in test tube and these process were repeated 6 times. After collecting the supernatant were placed in water bath for drying. The distilled water was added to the supernatant until its volume reached to 3 ml. 2 ml (Na 2 CO 3 ) of 20% were added in this solution. To this 0.5 ml Folin -ciocalteau regent was added and after 5 minutes more addition of 2 ml (Na 2 Co 3 ) from 20% Na 2 Co 3 solutions. The solution were mixed homogenously and then the test tube were brought in to the water bath in boiling water. At 650 nm their absorbance were checked. The Catechol was used as a standard .
Quantification of total alkaloids:-5 gm of the all the three extracts was weighed in a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was added and covered than allowed for 4 hours to stand. Extracts was filtered and was concentrated on a water bath to one-quarter of the original volume. Until the precipitation was completeDrop wise to the extract concentrated ammonium hydroxide was added. The solution was allowed to settle and collected the precipitated and washed with dilute ammonium hydroxide and then filtered. The residue was the alkaloid, was dried and weighed .
Determination of total tannins:-500 mg of the sample was weighed in a 50 ml plastic bottle. Added 50 ml of distilled water and shacked for 1 hours in a mechanical shaker. In a 50 ml volumetric flask was filtered and made up to the mark. Into a test tube, 5 ml of the filtered was pipetted out and mixed with 2 ml of 0.1 M FeCl3 in 0.I N HCl and 0.008 M potassium Ferro cyanide. At 120 nmtheir absorbance was cheeked out .

Determination of total saponins:-
Into a conical flask 20 gm of each extracts were put and 100 cm3 of 20% ethanol, aqueous were added. Over a hot water bath for 4 hours the samples were heated with continuous stirring at about 55°C. The residue re-extracted with another 200 ml 20% ethanol, and mixture was filtered. At about 90°C through water bath the combined extracts were reduced to 40 ml. Into a 250 ml separatory funnel the concentrate was transferred and 20 ml of diethyl ether was added and vigorously shaken. 60 ml of n-butanol was added. With 10 ml of 5% aqueous sodium chloride the combined n-butanol extracts were washed twice. In a water bath the remaining solution was heated. The samples were dried in the oven to a constant weight after evaporation; the saponin content was calculated .

Pharmacological activities:-
Pharmacological activities was carried out in methanolic extracts of Berberis lycium plant.
Experimental animals:-Both sexes of the albino mice of weight about 25 -30 gm were brought from the animal house of National Institute of Health (NIH), Islamabad. The animals were supplied with adlibitum water and standard pellet diet.

Anti-inflammatory activity by carrageenan induced inflammation:-
The mice were divided into five groups. Groups 3rd, 4th and 5th were treated with methanolic extract of at the doses of 200, 400 and 600mg/Kg respectively. Mice in the group 1 were given normal saline 10ml/kg. The 2nd group was treated with standard anti-inflammatory drug Aspirin(75mg) 150mg/kg. Carrageenan (20%) was injected in the right hind paw of albino mice of group 2nd, 3rd, 4th and 5th. Carrageenan solution was prepared by dissolving 20% carrageenan in 80% distilled water. To the mice of group 3rd, 4th and 5th. Methanolic extract at the dose of 200, 400 and 600mg/Kg was injected respectively in to the hind paw checked the diameter. Left for some time after extract injection and then measured the paw diameter at 1hour interval up to 4 hours and was compared with standard drugs to note the low and high potential of inflammation (Akhter et al., 2009).

Evaluation of Analgesic Activity:-Acetic acid induced writhing test:-
The mice were distributed into five groups. Groups 3 rd , 4 th and 5 th were treated with methanolic extract ofBerberis lycium at the doses of 200, 400 and 600mg/Kg respectively. The 2 nd group was treated with standard drug aspirin 150mg/kg. One hour after the treatment, 20% (10ml/kg) of acetic acid solution was injected by intra peritoneal injection. Abdominal writhings were counted for 5 minutes. For analgesic activity 1cc acetic acid (20%) was injected in to group 2 nd , 3 rd , 4 th and 5 th of mice. Aspirin solutions was prepared by dissolving one tablet in 10 ml of water. The solution was injected into mice of group 2 nd and checked the number of writhing's per 5 minutes. To the mice of group 3 rd , 4 th and 5 th methanolic extract at the dose of 200, 400 and 600mg/Kg was injected respectively. The results of group 3 rd , 4 th and 5 th was compared to the group 2 nd for low or high analgesic potential. The % inhibition calculated by the following formula (Akhter et al., 2009).

Antibacterial Activity Crude extracts Antibacterial:-
To screen the antifungal activity of the selected medicinal plants chloroform, methanolic and ethanolic extracts the agar well diffusion method was used (Perez et al., 1990). With all crude extracts of plants the assay was performed.
Media for bacterial culture:-By dissolving 25 g/l in distilled water Luria Broth, miller medium was prepared. Media PH was adjusted to 7.0. 100 ml of LB broth was distributed in 250 ml flask and autoclaved. In flasks Bacterial strains were inoculated and kept at 37ºC in shaker incubator at 150rpm overnight. By dissolving 40 g of LB agar in 1 liter of distilled water LB Agar was prepared and pH was adjusted and autoclaved.
Inoculum Preparation:-Bacteria strains from 24-hour old culture in LB broth (Miller) of selected bacterial strains were mixed with physiological normal saline solution until a McFarland turbidity standard [10 6 colony forming unit (CFU) ml -1 ] was obtained. In LB Agar Medium then this inoculum was used to seed.
Agar PlatesPreparation:-At room temperature LB agar was left to cool, it was poured into sterilized petri plates before to solidify.The agar well diffusion method (Perez et al., 1990) was used. Using a sterile cotton swab cultures lawn of the test organisms were made on the agar plates.Using a sterile borer under sterile conditions five wells were made per plate.
Extract Preparation for activity:-In 1 ml of DMSO 20mg crude extracts of all plant samples were completely dissolved. Solution of a standard antibiotic (2 mg/ml of Cefotaxime) was used as positive control. Negative control was used pure DMSO.

Measurement of zone of inhibition and Pouring of test solution incubation:-
Using micropipette, 75 μl of plant samples solution were poured in labeled wells. Each of the labeled plate was provided with samples of extracts, as positive standard cefotaxime and as negative standard di methyl sulphoxide (DMSO) was used. At 37ºC incubation was done. After 24 h of incubation, the diameter of clear zones, showing no bacterial growth around each well was measured. Three times activity was repeated and average of zone of inhibition with standard deviation was calculated.
1155 Statistical analysis:-All the tests were performed as individual triplicate experiment. All the data are shown as mean ± standard error of mean (S.E.M., n = number of Experiments). The statistical analyses were obtained by the one way analysis of variance (ANOVA), followed by the Dennett's test where necessary. P<0.05 was considered Significant.

Result and Discussion:-
Phytochemical analysis:-In the present research work the phytochemical investigation of methanolic, ethanolic and chloroform extracts ofBerberis lycium and Pharmacological activities of methanolic extracts (Anti-inflammatory, analgesic activity) and antibacterial activities in methanolic, ethanolic and chloroform extracts was carried out.

Discussion:-
In the present research work the phytochemical investigation of methanolic, ethanolic and chloroform extracts ofBerberis lycium and Pharmacological activities of methanolic extracts (Anti-inflammatory and analgesic activity) and antibacterial activities in methanolic, ethanolic and chloroform extracts was carried out. Qualitative analysis of Berberis lycium was carried out for the detection ofalkaloid, flavonoids, carbohydrate, phlobatannins, glycosides, saponins, phenol, terpenoids, tannins, cardiac glycosides and proteins. The results showed that alkaloids, flavonoids, carbohydrates, phlobatannins, saponins, phenols, terpenoids, tannins, cardiac glycosides was found in methanolic and ethanolic extracts, while alkaloids, phlobatannins, glycosides and protein were found absent in the aqueous extracts. Flavonoids, carbohydrates, saponins, phenols and terpenoids were found present in in the rhizome methanolic and ethanolic extracts. Highest amount of flavonoids was found in the chloroform extract as (14.20 ± 0.15mg/ml) followed by Alkaloids (12.10 ± 0.15mg/ml), phenolics (10.45 ± 0.10mg/ml), Saponins (06.22 ± 0.14mg/ml) and lowest amount of Tannins was found in (04.60 ± 0.65 mg/ml). The flavonoids was found in highest amount in methanolic as (17.55 ± 0.10mg/ml), followed by phenols (13.25 ± 0.50mg/ml), Tannins (11.55 ± 0.30mg/ml), Alkaloids (10.05 ± 0.10mg/ml) and Saponins was found in lowest amount (08.40 ± 0.45 mg/ml  (Ayoola et al., 2008). These phytochemicals showed antimicrobial activity through different mechanisms. With proline-rich protein tannins have been found to form irreversible complexes (Shimada, 2006) resulting in the inhibition of cell protein synthesis. (Parekh and Chanda, 2007) reported that tannins are known to react with proteins to deliver the typical tanning effect which is essential for the treatment of ulcerated or inflamed tissues. Herbs that have tannins as their key components are astringent in nature and are used for treating intestinal disorders such as dysentery and diarrhea (Dharmananda, 2003).Tannins and their derivatives are phenolic compounds considered to be primary antioxidants or free radical scavengers (Barile et al., 2007). These observations therefore support the use of Berberis lycium in herbal cure remedies, thus suggesting that Berberis lycium has potential as a source of important bioactive molecules for the treatment and prevention of cancer. The presence of tannins in Berberis lycium supports the traditional medicinal use of this plant in the treatment of different ailments. Alkaloid was another phytochemicals constituent's observed in the extract of Pteris quadriaurita. One of the best common biological properties of alkaloids is their toxicity against cells of foreign organisms. These activities have been widely studied for their potential use in the reduction and elimination of human cancer cell lines (Nobori, et al., 1994). One of the largest groups of phytochemicals as alkaloids in plants which have amazing effects on humans and this has led to the development of powerful pain killer medications (Kam and Liew, 2002). (Just et al., 1998) shown the inhibitory effect of saponins on inflamed cells. Saponin was found to be present in Berberis lycium extracts and has supported the usefulness of this plant in managing inflammation. Flavonoids, another phytochemicals shows a varied range of pharmacological activities like anti-inflammatory, antimicrobial, analgesic, anti-angionic, cytostatic, antioxidant and anti-allergic properties (Hodek et al., 2002).Several reports are presented on flavonoid groups which showing high potential biological activities such as anti-inflammatory, antioxidant, antiallergic reactions (Thitilertdecha et al., 2008). In the 1159 crude extractsthe bioactive compounds such as flavonoids and tanninsphytochemicals were present. Yet, these phytochemicals compounds were inducing the antimicrobial and antioxidants activities. By fractionationthe amount of active constituents in the crude extractsmight be dilute or improved their concentrations (Anyasor et al., 2010).

Conclusion:-
The above results confirmed that Berberis lycium has better anti-inflammatory analgesic and antibacterial activity. The pharmacological activity of the Berberis lycium may be due to the presence of phytochemical constituents. Some of these compounds possess analgesic, anti-inflammatory, antipyretic and antifungal activities. Further studies involving the purification of the chemical constituents of the plant and investigation in the biochemical pathway may results in the development of a potent analgesic, anti-inflammatory, anti-pyretic and antifungal agent with low toxicity and better therapeutic index.