INTRODUCTION:

Louranthus europpaeus L. its belong to the family Loranthaceae⁽¹⁾. The extracts from this plant have rich source of active compounds. been used therapeutically against various diseases including cancer, arthritis, cardiovascular illness and also widely used in adjuvant cancer therapy⁽²⁾. Endophytes are microorganisms that include bacteria and fungi living within plant tissues without causing and immediate negative effects, endophytes have been found in every plant species examined to date and recognized as the potential source of novel natural products for exploitation in medicine, agriculture and industry⁽³⁾. Some of the endophytes are the chemical synthesizers in inside the plants⁽⁴⁾. Many of them are capable of synthesizing bioactive compounds that can be used for defense against human pathogens and some of these compounds have been proven useful for novel drug discovery. Natural antioxidants may have free-radical scavengers, reducing agents, complexes of pro-oxidant metals, quenchers of singlet oxygen etc.

Recently research has been increased considerably in finding natural occurring antioxidants for use in foods or medical products to replace synthetic antioxidants, which are being restricted due to their adverse reaction suchas carcinogenicity. Antioxidant constituents from natural resources possess multi-affectedness in their multitude and magnitude of activity and provide enormous scope in correcting imbalance⁽⁵⁾.

MATERIAL AND METHODS:

For the preparation of crude extract 500g of grinded materials of Louranthus europpaeus, leaves and fruit was sinking in methanol taken in round bottom flask for about 24 hours. Then the extracts in solvent form filtered (by watt’s man filter paper) to a new round bottom flask. The process of filtration was recurring 3 days using supplementary concentration of methanol.

Phytochemical Screening

Methanol extract were subjected to find the phytochemical such as alkaloids, saponins, flavonoids, phenol, glycosides, terpenoids, steroids, reducing sugar, tannin, emodin, fatty acid, anthocyanin, coumarin, starch and protein by using standard procedures.

Alkaloids:

Take 0.5g of the methanoic extract on a filter paper and add some drops of Dragendroff’s reagent (solution of potassium Bismuth Iodide). The sample was then detected for the occurrence of yellow precipitation⁽⁶⁾.

Saponins:

2ml of water was added to 2ml of plant extract in a test tube. Shake the test tube well and wait for frothing for the detection of saponins⁽⁶⁾.

Flavonoids:

Take 4ml of extract and add 1.5ml of methanol. Warm the mixture besides adding metallic magnesium at that time add 4-5 droplets of hydrochloric acid and proverb the coloration (red)^(6,7).

Phenols:

To the plant extract 2ml ethanol and few drops of ferric chloride solution were added and observed the coloration. Formation of bluish black color indicates the presence of phenols^(6,7).

Glycoside:

Add few drops of ferric chloride and concentrated sulfuric acid to the solution of the extract (in glacial acetic acid), and watch for reddish brown coloration at the junction of two layers and the bluish green color in the upper layer⁽⁶⁾.

Terpenoids and steroid:

Take 0.2g of extract and treated with 0.5ml of acetic anhydride and 0.5ml of chloroform. Then the concentrated sulfuric acid was added slowly. Red violet color denotes terpenoids and green bluish color indicates steroids⁽⁶⁾.

Tannins:

To 0.5ml of methanol extract solution, ml of water and 1-2 drops of ferric chloride solution was added. Blue color was observed for Gallic tannins and green black for catecholic tannins⁽⁶⁾.

Emodins:

2ml of NH4OH and 3ml of Benzene was added to the extract. Appearance of red color indicates the presence of emodin⁽⁷⁾.

Fatty acid:

0.5ml of extract was mixed with 5ml of ether. The solution was allowed for evaporation on filter paper and dried. The appearance of transparency on filter paper indicates the presence of fatty acids⁽⁸⁾.

Iodine test:

Crude extract was mixed with 2ml of iodine solution. A dark blue or purple coloration indicates the presence of the carbohydrate⁽⁷⁾.

Antibacterial assay:

Filter paper discs (6mm diameter) were prepared using a punch machine. Filter paper discs were sterilized in a dry heat sterilizer and kept in the refrigerator for further use. A lawn of each bacterial isolate was prepared on MHA plates using a sterile cotton swab from the inoculums showing growth of 0.5 McFarland standards. MHA plates were dried for 15 minutes in the laminar air flow cabinet. Three filter paper discs were placed one on top of other on dried MHA plates and Louranthus europpaeus extract (20μl) were added on each disc separately. ethanol alone was used in order compare the antimicrobial activity. Bacterial species Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella spp. All plates were incubated at 37°C for 18-24 hours and the zones of inhibition (diameter in mm) were measured on the agar surface.

GC-MS Analysis:

A Shimadzu 17A GC coupled with Shimadzu QP5050 A (quadruple) Mass Spectrometer (Shimadzu, Japan), equipped with EI and a fused silica column DB-5 (30m x 0.25mm i.d.) of 0.25µm film thickness was needed. The oven temperature at 50ºC for 5 minutes and then programmed from 50-280ºC for 40 minutes. Helium flow rate of 2ml/min, with the split ratio of 1:30 mode was used for sample injection of 1µl and ionization voltage of MS-analysis was run by EI technique at 70ev. The volatile oil constituents were identified by matching their MS and retention index data with those of the standards ethnic spectra and by matching their fragmentation pattern in Mass Spectra with those of WILEY 139.LIB and NIST 12.LIB⁽³⁾. The retention indices were calculated by Kovats's procedure⁽⁸⁾.

Free radical scavenging activity (DPPH assay):

DPPH free radical scavenging activity is one of the basic reproducible methods for antioxidant screenings of numerous compounds. Free radical scavenging potentialities were tested against the methanolic solution of DPPH. DPPH accepts an electron or hydrogen radical to become a stable, diamagnetic molecule. It can be oxidized only with difficulty and irreversibly. Because of its odd electron 1,1-diphenyl-2-picryl hydrazyl shows a strong absorption band at 517nm, its solution appearing a deep violet color. As this electron becomes paired off, the absorption vanishes and the resulting decolonization is DPPH and converts into 1,1-diphenyl-2-picryl hydrazine. The purple color of DPPH changes to yellow (decolonization), indicating the scavenging efficacy of added substance. The change in absorbance at 517nm has been used as a measure of antioxidant property⁽⁹⁾.

The assay was standardized using ascorbic acid as standard. The reaction mixture 4ml contained, 0.1ml of (100-600μg/ml) various concentration of Viscum extract samples in0.1M Phosphate buffer (pH 7.2), 3.9ml of DPPH (0.025gm/l) solution was added to all the above test tubes. Incubated at room temperature (in the dark), for 30 minutes. A blank determination with 0.1ml methanol solution instead of standard treated similarly was maintained. For control 0.1ml of methanol with 3.9ml of DPPH solution was used. The optical density was measured at 517nm using a spectrophotometer.

Scavenging activity of DPPH free radical in percent was calculated according to the equation.

Percent inhibition % = (A-B) x 100/A

Where:

A= Absorbance of control reaction.

B= Absorbance of test sample.

The % inhibition of observance was plotted against the sample or the standard concentration to obtain the amount of antioxidant necessary to decrease the initial concentration of DPPH to 50% (IC₅₀). IC₅₀ values were calculated from calibration curve, IC50 values are defined as the concentration of a test compound required to achieve half maximal inhibition, lower IC₅₀value indicates greater antioxidant activity.

Statistical Analysis:

All the measurements were done in triplicate and statistical analysis was performed by statistical software. All the data were expressed as ±S.E.M. Statistical analysis were determined using one way analysis of variance (ANNOVA).

RESULT AND DISCUSSION:

Alcoholic extract consist of different groups of active components such as phenolic compounds, flavonoids, Alkaloids, Terpenoids, Tannins, Proteins, carbohydrates, saponin, and comarine (Table 1). Flavonoids are secondary metabolites, ubiquitous in fruitiest of different groups of active components and vegetables, which protect cell from degradation, stress, and act as signaling molecules. Phytoalexins, detoxifying agents, reduce toxic effects and stimulants ⁽¹²⁾. Recently indicated that active components can be nutritionally helpful by triggering the production of natural enzymes that fight disease, such as cancers, heart disease, and age-related degenerative diseases ⁽¹³⁾.

Table 1 : Active component in viscum album extract

Result	Name of active component	No.
+	Reducing sugar	1
+	Tannins	2
+	Flavonoids	3
+	Glycosides	4
+	Tannine	5
+	Emodin	6
-	Starch	7
+	Comarine	8
+	Phenols	9
+	Proteins	10
+	Terpenoid	11
+	Comarine	12
-	Fatty acid	13

Evaluated the anti mycobacterial activity of Louranthus europpaeus:

Subspecies reported that methanoic inhibited the growth of Klebsiella aerogenes, Proteus sp., Escherichia coli and Pseudomonas aeruginosa⁽¹⁴⁾ determined the antimicrobial activities ethanol, they showed that extracts gave antimicrobial activity against both Gram positive and Gram negative ⁽¹⁵⁾.

We found that the extract at the highest concentration gave the highest anti microbial activity as shown: Klebsiella spp.˃ Pseudomonas aeruginosa˃ Staphylococcus aureus˃ Escherichia coli, as shown in Figure 1 ⁽¹⁶⁾.

In the present study, the phytochemical profile of Viscum album alcoholic extracts was evaluated using the GC-MS technique. The cytotoxic effect was also the mass spectrometry analysis of the alcoholic extract Figure (2) evidenced the presence of phytosterols, like β-sitosterol, a powerful antioxidant that lowers the serum cholesterol levels in humans ⁽¹⁷⁾ campesterol, stigmasterol and has stabilizing effects on phospholipids bilayer and possess a protective effect against cardiovascular diseases, and also on colon and breast cancer⁽¹⁸⁾. Olean-12-en-3-yl acetate, a terpenoids with powerful antimicrobial, anti diabetic and anti amylase inhibitory activities⁽¹⁹⁾, was the major compound identified in both extract β-amyrin (3β-hydroxy-olean-12-en-3-ol), a pentacylic triterpene of considerable interest for its pharmacological properties, was also identified in the methanoic extract. Lupeol, lupenyl acetate, terpenoids that possess neuro protective, antioxidant and anti inflammatory effects ⁽²⁰⁾, were also identified in the methanoic extract. Tetralin derivatives such as (1S,4S)-1,6-dimethyl-4-propan-2-yl-1,2,3,4-tetrahydronaphthalene 3,3-dimethyl-1,2,3,4-tetrahydro-1,2-naphthalene diol that possess antimicrobial, antiviral, anticancer properties⁽²¹⁾ were also evidenced in the alcoholic extract. Fatty acids composition was as following: oleic, palmitic, 9-tetradecenoic, pentadecanoic, and docosatetranoic acids. An important natural antioxidant, vitamin E was evidenced in the analyzed mistletoe young leaves and branches.

The Ethanol extract of Louranthus europpaeus showed characteristic absorption bands at 3387 cm -1 and 1056 cm -1 (C-O) for a hydroxyl (-OH) group 2926 cm -1 , 2860 cm -1 ( for C- H stretching), 1381 cm -1 (for C-H bending), 1701 cm -1 for carbonyl group (C=O) and at 1614 cm-1 for C=C group.Fig () showed that the leaves and latex of Calotropis gigantea species were found to have cardiac glycosides. The cardiac glycosides were identified as calotropogenin and calotropin⁽²²⁾ also confirmed that the leaf parts of Caltrops gigantean species showed the presence of cardiac glycosides such as calotropogenin and calotropin besides other organic compounds such as amino acids, chlorophyll, amides, lignins, carbohydrates and starch pertaining to a healthy plant.

DPPH assay is one of the most widely used methods for screening antioxidant activity of plant extracts. DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become stable diamagnetic molecule. The reduction capability of DPPH radical was determined by the decrease in its absorbance at 517 nm, which is induced by different antioxidants. The decrease in absorbance of DPPH radical caused by antioxidants because of the reaction between antioxidant radical by hydrogen donation (23). It is visually noticeable as a change in colour from purple to yellow. The percentage of inhibition values of concentrated Viscum extract gave the highest inhibition percentage 86.04% in compare with the other concentration with IC₅₀ value of (50.32μg/ml).

Figure 4: Inhibition of Free radical formation (DPPH) of extract.

CONCLUSIONS:

It is concluded from the result that of Viscum album is composed of comparatively great amount of phytochemicals such as alkaloids, flavonoids, saponins, steroids, glycosides and phenols etc. These antimicrobial bioactive components mark the tested plant a confident candidate for future research and for medicinal usages.

ACKNOWLEDGMENT:

This work was supported by Mustansiriyah University, the authors would like to introduce their gratefulness to all the staff that helps to achieve this article.

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Received on 11.09.2019 Modified on 23.10.2019

Research J. Pharm. and Tech 2020; 13(6):2634-2638.

DOI: 10.5958/0974-360X.2020.00468.0