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GC-MS, HPLC Profiling, Antioxidant, Antimicrobial and Cytotoxicity Studies of Malcolmia Africana Leaves.

Byline: Tanveer Hussain Bokhari, Nasir Rasool, Maryam Riaz, Muhammad Riaz, Saira Hina, Rasool Buksh Tareen, Iftikhar Hussain Bukhari, Muhammad Zubair, Shazia Anwer Bukhari and Muhammad Shahid

Summary: Plants are known to be the richest source of natural antioxidant and antimicrobial properties. The use of herbs and medicinal plants as the first medicines is a universal phenomenon. The present study was carried out to examine the antioxidant, antimicrobial and cytotoxicity potential Malcolmia africana leaves extract, fraction, essential oil and fixed oil. The whole plant was extracted with absolute methanol and further fractionated with increasing polarity based absolute solvents. Different fractions were taken by solvent extraction method and their antimicrobial activities were determined. The IC50 and% inhibition by linoleic acid oxidation was evaluated for the antioxidant studies. The cytotoxicity of theplant extract and fractions were assayed against human blood erythrocytes (RBCs). The DPPH scavenging and linoleic acid oxidation assays were carried out. Qualitative and quantitative analysis of secondary metabolites was also carried out. The presence of phenolics was also studied by HPLC. The GC-MS analysis of Malcolmia africana essential oil and fixed oil was also carried out.

Key words: HP-LC, GC-MS, Malcolmia africana, Cytotoxicity, Antioxidant, Antimicrobial, Secondary metabolites

Introduction

Plants are known to be the major foundation of naturally occurring products, many of which can be utilized as agrochemical, pharmaceutical as fragrant, for flavoring ingredients, food additives purpose, and pesticides. It is usually well known that the most of the compounds are formed naturally, rather than synthetically, and they will be biodegraded more easily and consequently be more environmentally satisfactory [1]. Many products are secondary metabolites and can be obtained from direct extraction method from plants source which are growing in naturally occuring habitat, but many factors are present which can alter and affect their yield. Antibacterial, antiviral natural antioxidants, cytotoxic, fungicidal agents and nutrients have attained distinction in recent years; optimistic Fig and utilization among consumers are spreading day by day. In recent years, regular drug confrontation has been urbanized in both plants and human pathogenic microorganisms because of the informal use of antimicrobial drugs usually used in the treatment of contagious diseases [2]. As per earlier reports some authors have evaluated nutritional, antioxidant and biological studies of various plants of Pakistan [3-7] but more research is needed for the un explored plants. Several studies have exposed that medicinal and aromatic plants are the major sources of variousnutrient and non- nutrient molecules, many of these demonstrate antimicrobial and antioxidant assays which can protect the human body alongside both pathogens and cellular oxidation processes. Malcolmia africana is African mustard which is recognized as an annual, dicotyledonous type of herb, which belongs to the family Brassicaceae. It is dispersed in all over North Africa, Sothern Europe, central and southwest Asia and China and is renowned as a wild plant in fields of Khorasan province of saffron, Iran [8]. Malcolmia africana is annual herb with woody caudex. It has basal leaves with upright flower stalks, stiff. Height is about 18 inches. Stem is branched with hairs. Basal leaves are elongate and covered with hairs. Leaf surface is bumpy, and each bump has a forked hair. Flowers bloom during early spring. Flowers clustered at the top of the flower stalks, but the stalks continue to grow past the flower; petals 4, violet to pink. Fruits are erect, usually held away from the stem; to about2-inches long. Many tiny seeds per plants are present.

Various types of phytochemicals enclosed by Malcolmia species are: flavonoids, alkaloids, Phenolic acids contents, saponins contents, contents of tannins, contents of terpenoids, linolenic acid, and palmitic acid. Malcolmia africanawas selected todetermine the flavonoids contents [9]. Malcolmia africanais a cruciferous oil plant had high linolenic acid content with 57.37% used for industrial purpose [10]. Malcolmia africana was observed as forage and fodder [11]. Malcolmia africana was observed eaten readily by the hardy desert breed of sheep. In the desert-like region where frequent flocks of karakul sheep graze or browse all the year because of the chemical composition of M. africana and ascertained their nutritive value, as expressed in starch, protein and cellulose equivalents. Treculia africana (seed hull) was observed as feast as substitute for maize in diets for the African giant land snail [12]. The use of herbs and medicinal plants as the first medicines is a universal phenomenon. The present study was carried out to examine the antioxidant, antimicrobial and cytotoxicity studies of Malcolmia africana leaves extract, fraction, essential oil and fixed oil.

Results and Discussion

% Yield of Extracts and Fractions

The results of % yield of different extracts and fractions of leaves are shown in Table-1. The results of % yield are in the range of 0.12-4.16 g/100 g. The maximum yield was shown by absolut methanol extract of leaves (4.16 g/100 g). The n- butanol fraction of leaves also show good % yield (1.81 g/100 g). The less % yield was shown by ethylacetate (0.16 g/100 g). The decreasing order of% yield of extracts for leaves was: absolute methanolgreater than n-butanol greater than n-hexane greater than 80% methanol greater than aqueous greater thanchloroform greater than ethyl acetate.

Table-1: Percentage yield of extracts and fraction of leaves of Malcolmia Africana.

###Extracts and fractions###Percentage Yield

###n-Hexane###1.66

###Chloroform###0.16

###Ethyl acetate###0.12

###n-Butanol###1.81

###Absolute methanol###4.16

###Aqueous###0.30

###n-Hexane###1.66

Qualitative Phytochemical Analysis

Phytochemical screening exposed the presence of alkaloids, saponins, flavonoids, tannins, triterpenes, with no traces of steroid, glucoside, anthraquinones, phlobatanins in leaves. The phytochemical analysis of M. Africana is represented in Table-2. It endorses the presence of various phytochemicals in leaves. n-Hexane is non polar in nature and phytochemicals like alkaloids and flavonoids are polar in nature that's why none of the phytochemical was found in this fraction [13].

Quantitative Phytochemical Analysis

Quantitative analysis of leaves is shown in Tables-3 and 4 it expressed that alkaloid, flavonoid and saponins in n-hexane and n-butanol fraction were absent while alkaloids, flvonoids and saponins were present in metanolic fraction of leaves. In leaves methanol has maximum % of alkaloids (1.470.12) and flavonoid in leaves (0.800.2). In chloroform fraction of leaves only alkaloids (0.830.15) are present. Alkaloids were present in methanolic fraction of leaves. Maximum % of tannins was found in methanol fraction of leaves (11.220.08).

Total Phenolic Contents

The results of TPC were compiled in Table-5. The decreasing order of TPC values for leaves is: absolute methanol greater than chloroform greater than ethylacetate greater than n- butanol greater than aquous greater than 80% methanol greater than methanol greater than n- hexane. In leaves the methanolic extract (97.43) and chloroform (89.70) fraction showed greater amount of phenolic contents. The examined amount of ethylacetate (83.80) and n-butanol (70.37) was also of considerable importance. The n-hexane fraction for leave exposed minimum phenolic contents. The n-butanol and ethylacetate fractions indicated considerable phenolic contents in leaves.

Table-2: Qualitative analysis of the phytochemicals of extracts and different fractions of leaves of Malcolmia

Africana.

Phtochemicals###Sample Names for Leaves

###n-Hexane###Chloro-form###Ethyl Acetate###n-Butanol###AbsoluteMethanol###80% Methanol###Aqueous

###Alkalo

###ids

###-###-###-###-###+###+###+

###Saponins###-###-###-###-###+###+###+

###Flavonoids###-###-###+###-###+###+###-

###Tannins###+###+###+###+###+###+###+

###Terpenoids###+###+###+###-###-###-###-

###Steroids###-###-###-###-###-###-###-

###Glycosides###-###-###-###-###-###-###-

Anthraquinones###-###-###-###-###-###-###-

Phlobatanins###-###-###-###-###-###-###-

Table-3: Quantitative analysis of % of alkaloids, flavonoidsand saponins in Malcolmia Africana leaves of extracts and fractions.

Extracts, fractions and###Saponins

###Alkaloids % Flavonoids %

###essential oil###%

###n-Hexane###-###-

###Chloroform###-###-

###Ethyl acetate###-###0.400.2###-

###n-Butanol###-###-###-

###Absolute methanol###1.470.12###0.870.12###0.470.12

###80% Methanol###0.730.12###0.470.12###0.530.12

###Aqueous###0.930.12###0.670.12###600.20

Table-4: Quantitative analysis of % of TPC and tannins in Malcolmia Africana leaves of extracts and fractions.

###Extracts and fractions###TPC (%)###Tannins (%)

###n-Hexane###0.040.0007###1.600.02

###Chloroform###0.670.0031###6.70.12

###Ethyl acetate###2.150.0058###9.50.04

###n-Butanol###0.200.0013###2.910.04

###Absolute Methanol###2.540.0070###11.220.08

###80% Methanol###0.030.0007###3.990.05

###Aqueous###1.650.0050###1.510.01

Total Flavonoids Contents

The results of TFC are expressed in Table-5. TFC value for leaves was 5.30 (n-hexane) to150.18 (methanol) mg/100g. The decreasing order of TFC values for leaves can be: Absolute methanol greater than ethylacetate greater than n-butanol greater than methanol greater than chloroform greater than aquous greater than n-hexane. The reported values of TPC M. africana was, for leaves in the range of 5.83 (n- hexane) to 150.18 (methanol). The n-hexane showed the minimum quantity of flavonoids among all fractions. Ethyl acetate has 132.12 value of TFC for leaves so it has more TFC contents than other fractions after methanolic fraction.

Table-5: Total phenolic and flavonoid and total ascorbic acid contents of Malcolmia Africana leaves in extracts and fractions.

###TFC###Ascorbic

###Extract and###TPC (mg/100g

###(mg/100g###acid

###fractions###GAE)

###CE)###mg/100g

###n-Hexane###5.300.03###5.830.06###1.600.03

###Chloroform###89.700.11###15.470.13###4.000.05

###Ethylacetate###83.800.23###132.120.22###2.700.07

###n-Butanol###70.370.40###41.200.11###3.490.05

###Absolute

###97.430.24###150.180.52###3.790.06

###methanol

###80% Methanol###60.890.19###24.210.17###2.110.02

###Aqueous###73.931.03###15.500.10###4.770.04

DPPH Free Radical Scavenging Activity

The n-hexane and chloroform fractions of leaves showed maximum free radical sacavenging activity and lower IC50value. Results were compiled in Table-6. The order was found to be: n-hexane greater than chloroform greater than ethyleacetate greater than aqueous greater than 80%methanol greater than n-butanol greater than absolute methanol. The methanolic and n-butanol fraction showed minimum IC50 value.

Table-6: Antioxidant potential of Malcolmia Africana leaves in extracts and fractions.

###Extracts and fractions###IC50 (g/mL)###% inhibition

###n-Hexane###263.200.10###33.3330.524

###Chloroform###154.270.02###45.2740.228

###Ethyl acetate###73.370.07###53.4330.538

###n-Butanol###16.550.04###79.1540.971

###Absolute methanol###12.280.01###81.1590.849

###80% Methanol###18.500.05###53.1840.621

###Aqueous###39.180.04###44.8760.900

###BHT###9.01 0.07###84.9250.149

Percentage Inhibition of Peroxidation in LinoleicAcid System

In linoleic acid system the range of % inhibition in leaves. The maximum value of % inhibition was shown by BHT. The results are shown in Table-6. Order of decrease of % inhibition for leaves: Methanol greater than n-butanol greater than ethylacetate greater than 80% methanol greater than chloroform greater than aqueous greater than n-hexane. The maximum percentage inhibition was detected from methanolic extract (88.159) while n-butanolic fraction (79.154) also showed good inhibition. Other fractions also showed considerable inhibition.

Reducing Power

The absorbance increase with increase in concentration is shown by the results which are represented in Fig. 1 for leaves. All the fractions of leaves fulfilled the assay of reducing power and a linear increase to absorbance with concentration occurred. The maximum value of reducing power was given by ascorbic acid (Fig. 1).Total Ascorbic Acid Contents

TAAC results are shown in Table-5. TAAC values for extracts and fractions of leaves ranges from n-hexane (1.60) to chloroform (4.00).Antimicrobial ActivityAll the extracts and fractions of leaves wereexamined against four bacterial and three fungal strains. The results of antibacterial activity (zone of inhibition in mm) are shown in Table-7. The range of antimicrobial activity for leaves was 10.50 mm(ethyle acetate) to 17.63 mm (n-hexane). The n-butanolic (19.17 mm against Nitrospora) of leaves showed good activity. The results of antifungal activity are shown in 8. The standard used for antibacterial activity was Novidate and the standard used to check the antifungal activity was fungone.

Table-7: Antibacterial activity by zone of inhibition (mm) of Malcolmia Africana leaves in extracts and fractions.

###Extracts,

###Nitrospora

###fractions and###E.coli###B.cereus###S. epidermidis

###essential oil

###n-Hexane###-###15.150.02###17.630.25###-

###Chloroform###-###-###-###-

###Ethylacetate###10.500.20###-###-###11.650.03

###n-Butanol###17.770.15###12.570.02###17.080.02###11.850.04

###Methanol###14.700.20###-###8.670.03

80% Methanol###--###11.350.02###-###-

###Aqueous###--###12.370.02###13.360.02###-

###Novidate###18.750.75###21.770.49###25.260.36###22.92 0.43

Table-8: Antifungal activity by zone of inhibition (mm) by of Malcolmia Africana leaves in extracts and fractions.

###Extract and,

###A. niger###A. flavous###C. albecans

###fractions

###n-Hexane###9.230.21###-###8.910.10

###Chloroform###10.130.01###10.500.20###12.330.02

###Ethylacetate###8.770.01###9.250.02###-

###n-Butanol###-###9.50.1###9.540.01

###Methanol###-###-###-

###80% Methanol###13.150.02###9.930.02###-

###Aqueous###-###-###-

###Fungone###20.74 0.55###19.03 0.60###23.82 0.67

Minimum Inhibitory Concentration

The values of MIC were inversed to antimicrobial values. The results of MIC for bacterial strains are expressed in Table-9 and for fungal strain in Table-10. The MIC value disclosed the fact that greater the antimicrobial activity when MIC value is lower. The maximum haemolytic activity was shown by absolute methanol extract of leaves (8.1700.058). The n-butanol of leaves extract also exhibited substantial heamolytic activity about (7.3240.047). The minimum activity was shown by n-hexane fraction of leaves (2.3270.026). The results are expressed in Table-11. In our study, the percent lysis of human erythrocytes resulted in less than 5.0 % for all samples, thus these findings indicate minorcytotoxicity of the tested samples which was also comparable with earlier findings [14, 15].

Table-9: Minimum inhibitory concentration (MIC) against bacterial strain by Malcolmia Africana leaves extracts and fractions

Extracts and###S.

###E. coli###B. cereus###N. spora

fractions###epidermidis

n-Hexane###--###5.030.01###3.290.01###--

Chloroform###--###--###--###--

Ethylacetate###12.320.02###--###--###8.520.03

n-Butanol###3.030.02###7.770.02###4.020.02###8.040.02

Absolute Methanol 6.770.02###--###--###17.130.02

80% Methanol###--###8.960.02###--###--

Aqueous###--###7.920.02###7.170.02###-

Novidate###23.720.66###21.770.49

###5.26

###22.92 0.43

###0.36

Table-10: Minimum inhibitory concentration (MIC) against fungal strain by of Malcolmia Africana leaves in extracts and fraction

###Extracts and

###A. niger###A. flavous###C. albicans

###fractions

###n-Hexane###13.980.01###--###11.060.01

###Chloroform###12.130.02###12.370.02###7.710.06

###Ethylacetate###16.950.01###11.770.02###--

###n-Butanol###12.590.02###11.280.02###11.140.02

###Absolute methanol###--###--###--

###80% Methanol###6.590.01###13.970.02

###Aqueous###--###--###--

###Fungone###0.480.008###0.860.017###0.25###0.015

Table-11: % Haemolytic activity of extracts and fraction of Malcolmia Africana leaves.

###Extracts and fractions###% Haemolysis

###n-Hexane###2.3270.026

###Chloroform###3.7830.040

###Ethylacetate###2.8940.066

###n-Butanol###7.3240.047

###Absolute methanol###8.1700.058

###80% Methanol###3.6870.052

###Aqueous###2.3990.034

Antioxidant Potential of Essential Oil and n-HexaneExtract by Soxhlet

Essential oil of leaves showed less IC50 value and % inhibition than fixed oil. Results are shown in Table-12. The fixed oils of leaves showed IC50 (60.140.04).

Table-12: Antioxidant potential of essential oil and fixed oil Malcolmia Africana leaves.

###Type of Test###Samples###Essential oil###Fixed oil

###IC50 (g/mL)###Leaves###55.450.02###60.140.04

###% Inhibition###Leaves###40.7260.311###44.7260.311

Antimicrobial Activity in Zone of Inhibition (mm) ofEssential Oil and Fixed OilThe essential oil showed zone of inhibition (mm) (18.53 mm against Nitrospora). It showed good results and its minimum inhibitory concentration was found to be 3.220.02. Essential oil of leaves exhibited good results of antimicrobial activity against bacteria and fungus which represented that it

contained good phytoconstituents. The fixed oil of leaves exhibited good results against bacteria but did not give good results against fungal strains (Tables13-16).

Table-13: Antibacterial activity by zone of inhibition ZI (mm) of Malcolmia Africana.

Samples###E. coli###B. cereus###N. spora###S. epidermidis

Fixed oil###13.70.20###14.250.03###14.250.03###9.550.02

Essential oil###15.400.20###-###-###-###

Novidate###23.720.66###21.770.49###25.26 0.36###22.920.43

Table-14: Antifungal activity by zone of inhibition ZI (mm) by Malcolmia Africana fixed and essential oils.

Samples###Conc. (mg/mL)###A. niger###A. flavous###C. albicans

Fixed oil###50###-###9.530.15###12.530.02

Essential oil###50###-###-###-

Fungagone###50###20.740.55###19.030.6###23.820.67

Table-15: Minimum inhibitory concentration (MIC) (mg/mL) against bacterial strains by Malcolmia Africana fixed and essential oils

###S.

###Sample###E. coli###B. cereus###N. spira

###epidermidis

###Fixed oil###6.780.02###6.550.01###6.570.02###14.340.02

Essential oil###5.190.01###-###-###-

###Novidate###0.460.021###0.970.013###0.390.007###0.420.015

Table-16: Minimum inhibitory concentration (MIC) (mg/mL) against fungus strains by Malcolmia Africana fixed and essential oils

###Samples###A. niger###A. flavous###C. albicans

###Fixed oil###-###11.350.02###7.130.02

###Essential oil###-###-###-

###Fungone###0.480.008###0.86 0.017###0.250.015

HPLC Analysis of Different Extracts and Fractions ofM. africanaLeaves showed five acids (caffeic acid, syringic acid, chlorogenic acid, vannilic acid and gallic acid). Results were expressed in Table-17.

Table-17: HPLC Analysis of different fractions of Malcolmia Africana leaves for phenolic contents

###Chloroform###Ethyl acetate###n-butanol

Phenolic acids

###(ppm)###(ppm)###(ppm)

###Gallic acid###-###0.47###-

Caffeic acid###3.63###133.50###-

p-coumeric acid###-###-###-

Syringic acid###3.52###-###0.23

Chlorogenic

###14.27###94.30###-

###acid

Vanillic acid###-###-###-

GC-MS Analysis of Essential Oil Fixed Oil

Chemical composition of essential oil of M. africana leaves were identified by GC-MS analysis,shown in Table-18. The results indicated that 2, 2- dimethoxybutane is present in leaves having percentage composition 0.46 respectively. Similarly Linolenic acid is also present in fixed oil fraction of leaves having % age composition of 0.79%. Eicosanoic acid was also identified in leaves (21%). In leaves lupenyl acetate (5.31), Palmitic acid (1.08), Tetradecanoic acid (1.43), Clionasterol (18.63), Pentadecane (1.37) were identified. Linolenic acid (0.79%) has previously been identified in M. africana (PENG et al., 1997).

Table-18: Chemical composition of essential oil of Malcolmia Africana leaves as identified by GC-MS analysis

Retention time###%

###Name of the compound

###(min)###Compsition

###3.15###1-Pentanol###1.11

###3.42###2,2-Dimethoxybutane###2.67

###7.9###Undecane###0.71

###10.46###Pentadecane###1.37

###12.84###Tetradecanoic acid###1.43

###12.96###Lupenyl acetate###5.31

###13.81###Palmitic acid, methyl ester###1.08

###13.9###Linolenic acid###0.79

###13.99###Eicosanoic acid###21.00

###9,12-Octadecadienoic acid,

###14.72###1.22

###methyl ester

###14.95###2-Octylcyclopropene-1-heptanol###22.03

###15.04###Octadecanoic acid###1.88

###16.34###Lupenyl acetate###7.25

###16.62###Stigmasterol###3.26

###16.8###Diisooctyl phthalate###4.05

###17.32###Clionasterol###18.63

###19.09###Olean-12-en-3-yl acetate###3.55

Table-19: Chemical composition of fixed oil of Malcolmia Africana leaves as identified by GC-MS analysis.

Retention time###%

###Name of the compound

###(min)###Compsition

###3.42###2,2-Dimethoxybutane###0.46

###11.25###2,4-Di-tert-butylphenol###0.39

###11.8###9-Eicosene###0.39

###12.83###Tetradecanoic acid###0.6

###13.05###1-Nonadecene###0.43

###Hexadecanoic acid, methyl

###13.8###4.28

###ester

###Ascorbic acid 2,6-

###14###15.37

###dihexadecanoate

###6-Octadecenoic acid, methyl

###14.75###9.95

###ester

###14.86###Stearic acid, methyl ester###1.43

###14.96###Cis-9-Hexadecenal###38.95

###15.04###Eicosanoic acid###6.74

###15.7###Tetracosane###0.4

###15.89###N,N-Dimethylcholestan-6amine###0.63

###16.9###1-Bromotetracosane###0.43

###17.5###Hexatriacontane###0.73

###Tetracosanoic acid, methyl

###17.66###0.35

###ester

###17.99###1-Pentatriacontanol###0.45

###18.12###Trans-Squalene###1.9

GC-MS analysis of chemical composition of leaves fixed oil extracted by soxhlet apparatus, of M. africana was done and different type of chemical

compounds were found shown in Table-4.19. 2, 2- dimethoxybutane was found in leaves 0.46%. The % composition of some important phytoconstituents such as eicasanoic acid (6.74%), 6-Octadecenoic acid, methyl ester (9.95%), Hexadecanoic acid, methyl ester (4.28%), Ascorbic acid 2,6- dihexadecanoate (15.37%), Trans-Squalene (1.90%). During review of literature it was found that some phyto-components, methyl ester showed biological activities [16].

Experimental

Chemicals and Reagents

Gallic Acid, 2,2-diphenyl-1-picrylhydrazyl radical (90.0 %), Folin-Ciocalteu reagent, butylatedhydroxytoluene (99.0 %), Linoleic acid, ascorbic acid, aluminum chloride, ferric chloride, ferrous chloride, sodium nitrite, trichloro-acetic acid, potassium ferricyanate were purchased from Sigma Chemicals Co (St, Louis, MO, USA).All other analytical grade chemicals such as ammonium thiocyanate, methanol and anhydrous sodium carbonate ferrous chloride were obtained from Merck (Darmstadt, Germany).

Plant Collection

Malcolmia africana leaves were selected because negligible work was done on this plant but intensive review and ethno pharmacological information was available on its genus and species. The selected plant was in herb form collected from the Quetta and Ziarat valley and was identified by Dr. Rasool Bukhsh Tareen Department of Botany, University of Balochistan, Quetta Pakistan. A voucher specimen (MA-RBT-04) was deposited in the herbarium/collection Department of Botany, University of Baluchistan, Quetta, Pakistan.

Plant Extracts/ Fractions Preparation

The leaves of Malcolmia africana were washed with cold water to remove dust and other extraneous matter. The shadedleaves were grinded into fine powder by using commercial blender. Extract of selected medicinal plant Malcolmia africana was prepared in methanol and next fractions were prepared by n-Hexane, Chloroform, Ethyl acetate and n-Butanol. This powder of leaves weighed (670 g) and added to glass jar and dipped into methanol to get extract at room temperature for seven days with two times daily shaking for five min. Residues of leaves were suspended separately into100 mL distilled water and fractioned with different solvents on polarity base with n-hexane and chloroform ethyl acetate and n-butanol respectively.

After this aqueous residue was saved and different activities were carried out on these fractions. The extract and fractions were stored at 4C until used for analysis.

Phytochemical Analysis

Powdered leaves of the plants were subjected to qualitative and quantitative phytochemical screening to analyze the presence of alkaloids, tannins, spaoninsand steroids by using standard phytochemical procedures [17].

Evaluation of Antioxidant Activity of Malcolmia africana

The amount of total phenolic was determined using Folin-Ciocalteu reagent [18, 19]. The amount of total flavonoids was examined spectrophotometerically, as previously reported by [20]. 1, 1-diphenyl-2-picrylhydazyl radical (DPPH) scavenging assay of fractions and n-hexane extract by soxhlet of was assessed spectrophotometerically as reported earlier by [21]. Per oxidation in the terms of% inhibition was evaluated to estimate the antioxidant assay of all the fractions, essential oil and n-hexane extract by soxhlet in Linoleic acid system followed the the method described by [22]. The reducing power of the extracts, n-hexane extract by soxhlet was determined according to the reported procedure followed by [23] with little amendment. Ascorbic acid contents were estimated in different parts of plant extracts and fractions as per the reported method used by [24].

Antimicrobial Assay of Plant Extract and its DifferentFractions

Microbial Strains

The antimicrobial activities of the leaves extract and its all fraction were tested against a panel of microorganism, including four strains of bacteria i.e. Bacillus cereus, Escherichia coli, Nitrospora, S. epidermidis and three strains of fungi i.e. Aspergillus niger, C. albicans, A. flavous. These pathogenic strains were used to determine the antibacterial activities with reference to the minimum inhibitory (MIC) of the leaves extract the selected and fractions of selected medicinal plant.

Antibacterial Assay

Bacterial Growth Medium, Culture and InoculumsPreparation

Pure cultures of bacteria strains were maintained on nutrient agar medium in the slants and Petri plants. For the inoculums preparation 13g/L of nutrient broth (Oxoid) were suspended in distilledwater, mixed well and distributed homogenously. Autoclaved 10L of pure culture of each bacterial strain as mixed in medium and placed in shaker for24 h at 37C.

Antibacterial Assay by Disc Diffusion Method

Nutrient agar (Oxoid) 28 g/L was suspended in distilled water, mixed well and distributed homogenously. Before the medium was transferred to sterilized petri plates; inoculum (100 L/100 mL) was added to the medium and poured in sterilized petri plates. Sterilized the medium at 121C for 15 min in autoclave. After this, small filter paper discs were laid flat on growth medium containing 100 L of extract. The petri plates were incubated at 37C for24 h, for the growth of bacteria. The extracts having antibacterial activities were inhibited the bacterial growth and clear zones were formed. The zones of inhibition were measured in millimeters using zone reader [25].

Antifungal Assay

Fungal Growth Medium, Cultures and InoculumsPreparation

Pure culture of fungal strains was maintained on potato dextrose agar medium in the slants and Petri plates. For the inoculums preparation21 g/L of mullerhinton broth (Oxoid) was suspendedin distilled water, mixed well and distributed homogenously. Autoclaved 10L of pure culture of each fungal strain was mixed in medium and placed in shaker for 24 h at 37oC. The inocula were stored at4oC.

Antifungal Assay by Disc Diffusion Method

Potato dextrose agar (Oxoid, UK) 28 g/L was suspended in distilled water, mixed well and distributed homogenously. The medium was sterilized by autoclaving at 121C for 15 min at pressure (15 Id/sq. inch). Before the medium was transferred to Petri plates, inoculums (100L/100mL) was added to the medium and poured in sterilized Petri plates. After this, small filter paper discs were laid flat on growth medium containing 100 L of extract. The Petri plates were than incubated at 28C for 48 h, for the growth of fungus. The zones were formed around the discs. The zones of inhibition were measured in millimeters using zones reader [25,26].

Minimum Inhibitory Concentration (MIC)

Resazurinmicrotiter-Plate Assay

The minimum inhibitory concentration of extracts and different fractions of M. africana was evaluated by a modified resazurinmicrotitre-plate assay as reported [27].

Haemolytic Activity

Haemolytic activity of plant extraction and its fractions was checked by the literature method used by [14, 26]. 0.1% titron X-100 was taken for each assay as a positive control and phosphate buffer saline (PBS) was taken for each assay as a negative control. The absorbance was noted at 576 nm with micro plate reader (Bio Tec, USA).

Preparation of n-hexane Extract by Soxhlet

By following the reported method of [28,29] with little modification, n-hexane extract was prepared by using Soxhlet and weighed 20 g. For further analysis the extract was stored in vial.

Sample Preparation for HPLC

By following the reported method of [30] with small modfication, different fractions of phenolic extracts n-butanol, ethyl acetate and chloroform of leaves of M.africana (0.05 g of each fraction) was taken and then 20 mL of respective solvent was added in it. In a refrigerated incubator shaker (NJ, USA), the mixture was shaken for 60 min at room temperature. In a universal 320r Hettich Zenrifug (Tuttlingen, Germany), the mixture was centrifuged at 9000 rpm at 4C for 5 min, for the further estimation of phenolic compounds of the supernatant.

Conclusion

It is concluded from discussion and results that the leaves of M. africana exhibited considerable antioxidant and antimicrobial activities against bacterial and fungal strains. The cytotoxicity studies by haemolytic activity showed that samples have less than 10% lysis for RBCs. In HPLC analysis of different fractions also exhibited prence of phenolics. Essential oil and n-hexane fraction by soxhlet of plant showed prence of phytoconstitunts. Hence, it is said that the leaves of Malcolmia africana might be used as herbal medicine.

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Publication:Journal of the Chemical Society of Pakistan
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Date:Jun 30, 2014
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