Analysis of Phytoconstituents and Biological Activities of Different Parts of Mahonia nepalensis and Berberis aristata

The phytochemicals and biological activities of extracts from leaves and stem of Mahonia nepalensis and Berberis aristata were carried out. Phytochemical screening showed the presence of alkaloids, steroids, polyphenols, quinones, glycoside, flavonoid, terpenoid and cardiac glycoside in the hexane, ethyl acetate and methanol extracts of leaf and stem of these two plants. The column chromatography of methanol extract of stem of Mahonia nepalensis resulted in isolation of four pure compounds MN1, MN2, MN3 and MN4. Out of four isolated compounds, two were identified as MN1:sitosterol and MN2: Berberine by comparison of melting point, Co-TLC, IR and UV spectra of authentic sample. Potent pharmacological activity of Mahonia nepalensis and Berberis aristata were revealed from antimicrobial activity and brine shrimp bioassay. Methanol extracts of stem of Mahonia nepalensis and Berberis aristata showed significant zone of inhibition of 18 mm and 21 mm respectively against the Staphylococcus aureus. Methanol extract of Berberis aristata were comparatively little stronger against Staphylococcus aureus than methanol extract of Mahonia nepalensis. LC50 values (μg/ml) of methanol extracts of stem of Berberis aristata and Mahonia nepalensis were found to be 8.058x10-4 and 8.3 whereas methanol extracts of leaf of Mahonia nepalensis and Berberis aristata were 389.04 and 1303.166 respectively.


Introduction
Nepal is well known for superb collection of medicinal and aromatic plant resources that grow luxuriously in tropical forests to alpine meadow [1].Since past, parts of these medicinal plants or their extracts have been used as traditional medicine.Traditional medicines are safe, easily accessible and affordable form of health care for much of the rural population in Nepal.Plants used in traditional medicine are important sources of novel biomolecules with application for the manufacture of pharmaceuticals and cosmeceuticals [2].Berberidaceace is a family of shrubs and herbs, mainly of the Northern temperate zone, with simple, pinnate, or peltate leaves with spines.There are 14 genera in this family, and Mahonia and Berberis are among them.Mahonia is readily distinguished from Berberis by its compound leaves, spineless stems and inflorescence of several dense spikes.Berberis is distinguished by its undivided spiny toothed leaves and its spiny stems with yellow wood [3].Mahonia nepalensis belongs to the family Berberidaceae and is known vernacularly as "Jamanemandro" in Nepali and "Michiki swan" in Newari.It is medium sized fully hardy perennial evergreen shrub with yellow flowers in winter.This shrub has an ultimate height of 6m /19.7ft.Its origin is in Nepal.It is widely distributed in the high mountainous areas at altitude of about 1000m -2000m in Nepal, Sikkim, Bhutan, China, Vietnam, etc.It is useful in architectural and security barrier in garden, traditional essential flower for conducting Bel Bibaha and Bratabanda in Newar community.The stem and wood of this plant have antiinflammatory, anti-bacterial, anti-fungal activity.It is particularly used for the treatment of skin diseases like eczema, psoriasis, etc.This plant contains alkaloids as the major compounds which belong to class protoberberines and bisbenzylisoquinolines [4] Berberine [5], Jattrrorzhine, O-methyl puljabine [6], Isotetradine, Homoaromaline etc. were isolated from the stem of this plant [7].Berberis aristata also belongs to Berberidaceace family and is commonly known as "Chutro" in Nepali, "Daruhaldi" in Hindi and "Indian Berberry" in English.It is spinous herb native to northern Himalaya region, widely distributed from Himalayas to Sri Lanka, Bhutan and hilly areas of Nepal.It is used in ayurvedic medicine from very long time.The plant is used traditionally in inflammation, diarrhea, wound healing, skin disease, menohrrhagia, jaundice, and affection of eyes.A very valuable ayurvedic preparation" Rashut" is prepared from this plant [5].It is useful in treatment of jaundice, diabetes, cancer, malaria etc. and has good anti-oxidant property, anti-pyretic activity, analgesic activity, anti-fungal activity and anti-microbial property, anti-inflammatory, anti-platelet activating factor were isolated from its stem while citric acid, malic acid from fruit and E-caffeic acid, quercetin, chlorogenic acid, meratin and rutin from flower have also been isolated from this plant [15].Due to their important pharmaceutical importance, we have tried to isolate the plant metabolites and determine the photoconstituents of different parts of these two valuable plants and studied their biological properties, isolated some of the important compounds and tried to characterize and identify the isolated compound as well.

Sample collection and extraction
The stem and leaves of Mahonia nepalensis and Berberis aristata were collected from Bhaktapur and Palpa district respectively in April, 2013 and thoroughly dried in shade.About 50 g of stem and leaves of the two plants were ground to fine powder.The grinded parts were then successively extracted with hexane, ethyl acetate and methanol on the basis of their increasing polarity by using Soxhlet apparatus.The extracts were concentrated using rotatory evaporator and left for removal of solvent.After the solvents were completely removed, they were used for different purposes like phytochemical screening, biological activities study and isolation of chemical constituents.

Phytochemical screening
A small amount of dry extracts of plant materials were subjected to phytochemical screening.The method employed was based on the standardized procedure with slight modification [16][17][18].

Test for Tannin/Polyphenols
To a portion of extract diluted with water, 3-4 drops of 10% FeCl3 is added, a blue color was observed for gallic, tannins and green color for catecholic tannin.

Test for Reducing Sugar
To 0.5ml of plant extract, 1ml of water and 5-8 drops of Fehling's solution was added and heated over water bath.Brick Red precipitate indicated the presence of reducing sugar.

Test for Quinines
To the extract, Freshly Prepared FeSO4 Solution (1ml) and Ammonium Thiocyanate (few crystals) were added and treated with conc.H2SO4 drop by drop.The deep red color was persistent indicating the presence of quinines.

Test for Glycosides
For testing the glycosides, the protocol was slightly modified.To the extract, 5ml Molisch's reagent was added and conc.H2SO4 was added drop wise without disturbing the solution.A violet ring at the junction of two liquids was observed and on shaking the solution turned violet completely indicating the presence of glycosides.

Test for Flavonoid
i. Shinoda test: 4ml of extract solution was treated with 1.5ml of 50% methanol solution.The solution was warmed and metal magnesium was added.To this solution, 5-6 drops of conc.HCl was added and red color was observed for flavonoid and orange color for flavones.ii.5ml of dilute NH3 solution was added to the aqueous filtered solution of each fraction followed by the addition of conc.H2SO4.The appearance of yellow color indicated the presence of flavonoid.The yellow color disappeared after some time.

Test for Terpenoid
About 0.2g of each sample was mixed with 2ml of chloroform first and 3ml of conc.H2SO4 was added to each mixture.The formation of a reddish brown coloration at the interface indicates the presence of terpenoid.

I.
Meyer's Test: To 2ml of extract, 1ml of Meyer's reagent (picric acid solution) was added.White precipitate and pale yellow precipitate indicates presence of alkaloids.ii.Dragendroff's Reagent Test: 2ml of extract and each fraction were warmed with 2%.H2SO4 for 2min.After filtration of the reaction mixture a few drops of Dragendroff's reagent were added to each filtrate.Orange red precipitate indicates presence of alkaloids.

Test for Saponins
About 2g of powdered sample was boiled in 20ml of distilled water in a water bath and filtered.10ml of filtrate was mixed with 5ml of distilled water and shaken vigorously.The appearance of frothing indicated the presence of saponins.

Test for Volatile oils
2ml extract was shaken with0.1ml of NaOH and a small quantity of dil.HCl.A white precipitate was formed which indicated presence of volatile oils.

Test for Cardiac Glycosides
5ml of plant extract was treated with 2ml of glacial acetic acid containing one drop of FeCl3 solution.A brown ring at the interface indicated a deoxy sugar characteristic of cardenolides.A violet ring was appeared below the brown ring, while in acetic acid; a greenish ring was formed just gradually throughout thin layer which showed the presence of cardiac glycosides.

Test for Steroids
1gm of plant extract was dissolved in few drops of acetic acid.It was gently warmed and cooled under tap and a drop of conc.H2SO4 was added alongside of tube.Appearance of green color indicated presence of steroids.

Separation of compound by using column chromatography
23.2g methanol extract of stem of Mahonia nepalensis was adsorbed in 20.07g silica gel (60-120) mesh and loaded in the column having diameter 3cm and length 60cm packed with150 mg activated silica gel (60-120)mesh.The 40.5cm long column was eluted with gradient of hexane, ethyl acetate, and methanol to obtain no. of fractions which resulted in isolation of compound MN1, compound MN2, compound MN3 and compound MN4.Compound MN1was obtained on concentration of fraction (25-27) in rotator evaporator while Compound MN2 from fraction (190)(191)

Antimicrobial tests
The extracts of Mahonia nepalensis and Berberis aristata were screened for their antimicrobial activity i.e. determination of zone of inhibition against tested organism by Agar well diffusion method [

Brine Shrimp Bioassay
Brine shrimp Bioassay was performed using standard procedure.Brine shrimp eggs were hatched in artificial sea water.The Brine Shrimp larvae were cultured under prescribed laboratory condition [20] and used against methanol extracts.The number of survived shrimps was counted and LC50 value was calculated.

Isolation of Plant Metabolites
The different parts of plants specially leaf and stem selected on the basis of their medicinal use in Ethno medicine were successively extracted on the basis of increasing polarity.The yield of these extract and their physical characteristic were shown in (Table 1).The highest yield % was observed for the methanolic extract of stem of B.aristata and was found to be 22.6 while the yield % for methanolic extract of stem of Mahonia nepalensis

Phytochemical Screening
The phytochemical screening of all plant materials were done on the basis of the procedure given by Alamzeb K (2013), Talukdar and Chaudhary (2010), and Prof. I. Culie (1990) [16][17][18].The results obtained are given below in Table 2.

Biological activities of plant extract The antimicrobial susceptibility test
The study involved the antimicrobial activity tests of the different extracts.Different bacteria were used for the test of the activity of the extracts.The results of the qualitative antimicrobial screening of different extracts were as shown in (Table 3).

Comparative Study of anti-microbial activity
The results of the antimicrobial susceptibility test of the different extracts showed that the crude hexane extracts and ethyl acetate extract of stem and leaf of M. nepalensis were resistant to all of the bacteria species tested while ethyl acetate extract of B. aristata was found to be moderately active against Staphylococcus aureus while hexane extract of stem of B. aristata was slightly active against S. aureus.But the methanol extracts of stem of both plant species were found to be strongly active towards the gram positive bacteria Staphylococcus aureus.The methanol extract of B. aristata showed same pharmacological effect of Nalidixic acid control while methanol extract of M. nepalensis showed little lower pharmacological effect than that of control.Hence both of these methanol extract of M. nepalensis and B. aristata were pharmacologically active against Staphylococcus aureus while they were resistant to bacteria Kleibsella pneumonia.The methanol extract of leaf of both plant species were also found to be resistant to above bacteria species.DMSO was used as solvent control and it showed no effect.It was also found that Salmonella typhimurim and Salmonella typhimurium strains of bacteria were resistant to Nalidixic acid (Data not shown)

Brine Shrimp Bioassay
For the study of biological activity of plant material, the newly hatched brine shrimp nauplii were exposed to the plant extracts.The biological activities were evaluated on the basis of their toxicities towards these nauplii.In this method, LC50 values (μg/ml) for different fractions were determined and those having less than 1000 are considered as pharmacologically active.The results obtained during this study are given in (Table 4).

Figure 9 :
Figure 9: Chemical structure of 7, 8-Dihydro-8methoxy Berberine Compound MN4: A needle shaped dark Orange brown colored crystalline compound MN4 was obtained in pure and dry state.A sharp melting

Table 1 :
Percentage yield of different plant extracts and their physical characteristics.Similarly the highest yield % of methanolic leaf extract of Mahonia nepalensis was 22.4 whereas the yield % of methanolic leaf extract of B.aristata was only 3.8%.Hexane extract were generally sticky and has comparatively low yield while ethylacetate extract yield was medium.

Table 4 :
cytotoxicity value of different plant extracts