Sulayman Abid, Saad Touqeer
Faculty of Pharmacy, The University of Lahore, Lahore, Pakistan
Keywords: Antibacterial; Antifungal; Plant; Extract; Activity; Phytochemistry; DPPH.

The present study was aimed to investigate the phytochemistry and to determine the antibacterial, antifungal and antioxidant activities of the methanolic extract of the two plants namely, Breynia disticha and Vernonia elaeagnifolia. Antimicrobial activity was determined using disk diffusion method whereas antioxidant activity was carried out using DPPH assay. Antimicrobial activity was absent in B. disticha whereas V. elaeagnifolia possessed antibacterial activity against P. mirabilis (9.0±0.2 mm) and antifungal activity against S. cerevisae (8.0±0.1 mm). Both plants possessed significant antioxidant activities (more than 90% scavenging) comparable to the standard drug BHT. The study proves that both plants have high medicinal value.

Article Information

Identifiers and Pagination:
First Page:152
Last Page:155
Publisher Id:19204159.7:3.2015
Article History:
Received:June 4, 2015
Accepted:June 26, 2015
Collection year:2015
First Published:July 1,2016



Microbial infections are one of the leading causes of death worldwide. The pathogenic microorganisms are becoming resistant to many of the commercially available antimicrobial drugs.  Efforts by scientists in the area of chemotherapy have been increased to a great extent in the last two decades [1,2]. Natural products can be a good source of obtaining highly safe, potent and low cost antibacterial and antifungal drugs.

Reactive oxygen species (ROS) are the metabolic by-products produced in the human body. These free radicals are very harmful to our body and can damage the cell membrane and nucleic acid of our body if produced in excess amount. In order to reduce the oxidative stress caused by various endogenous and exogenous free radicals, antioxidant compounds are used. These compounds react with free radicals and neutralize them. Most of the potent antioxidants currently available are capable of scavenging a variety of ROS in both in-vitro and in-vivo conditions [3].

Breynia disticha is a shrub belonging to the family Phyllanthaceae. It is commonly found in gardens and public parks. The leaves of the plant are multicolored with green, purple and white to be most prominent of all the colors. Vernonia elaeagnifolia is creeper and belongs to family Asteraceae. It is commonly grown in gardens and lawns for screening purposes. The plant has been traditionally used as a leech repellant [4,5,6].

Until present, no study has been carried out in order to determine the antimicrobial and antioxidant activity of the plants. The aim of the present work is to report the antibacterial, antifungal, antioxidant and free radical scavenging effect of the plants for the first time.


Plant material

The aerial parts of Breynia disticha and Vernonia elaeagnifolia were collected during the winter season from different parts of Lahore, Pakistan. The plants were identified by Dr. Ajaib Choudhary, Department of Botany, Government College University, Lahore. The voucher numbers received for B. disticha and V. elaeagnifolia were GC.Bot.Herb. 2282 and GC.Bot.Herb. 2283 respectively.



Preparation of extract

The plant material was shade dried and ground into coarse powder. It was extracted by cold maceration twice (7 days each) using methanol. The extracts obtained were dried using rotary evaporator and stored in air tight container in a refrigerator until further use.


Methanol of analytical grade was purchased from Panreac, Spain. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH), Butylated HydroxyToluene (BHT) and Dimethly sulfoxide (DMSO) were purchased from Sigma-Aldrich, USA. All chemicals used in the phytochemical tests were of analytical grade.

Microbial strains

The clinical strains of Klebsiella pneumonia and Proteus mirabilis were used in the antibacterial assay whereas the clinical strains of Aspergillus flavus and Saccharomyces cerevisae were used in the antifungal assay. All the microbial strains were kindly supplied by Institute of Molecular Biology and Biotechnology, University of Lahore.

Phytochemical studies

Phytochemical tests for the identification of alkaloids, glycosides, tannins, flavonoids and saponins were performed according to the method described by Raaman [7].

Antibacterial activity

Antibacterial activity was determined by disk diffusion method described by Saad et al., [8]. Bacterial strains cultured in nutrient broth were spread over the surface of prepared Mueller Hilton Agar (MHA) media in sterilized Petri dishes. Sterile paper disks (6.0mm diameter) were then placed and 20µl of sample (20mg/1000 µl DMSO) was applied. The plates were incubated at 37ºC for 24h and the diameter of zone of inhibition was recorded.

Antifungal activity

Antifungal activity was also determined by disk diffusion method [8]. The fungal culture prepared in normal saline was spread over the surface of Sabouraud Dextrose Agar (SDA) media. To the paper disks, 20µl of sample (20mg/1000 µl DMSO) was loaded and then kept at room temperature for 24h. The diameter of zone of inhibition was determined.

Antioxidant activity

The antioxidant activity of methanolic extracts of plants was determined by spectrophotometric method [9]. The extracts used in the assay were dissolved in methanol to obtain a concentration of 1mg/ml. To 1ml of the sample solution 3 ml of Methanolic solution of DPPH (0.1mM) was added and incubated at 27oC for 20min in order to complete the reaction. The absorbance was determined at 517nm. BHT was used as a standard drug and the assay was carried out in triplicate.


Percentage scavenging was calculated using the following formula:

% age scavenging = [Abs (control) – Abs (sample) / Abs (control)] x 100


The results of preliminary phytochemical study are given in table 1. All major secondary metabolites were found in Breynia disticha methanolic extract (BDME) except for alkaloids. In case of Vernonia elaeagnifolia methanolic extract (VEME) glycosides and saponins were found to be absent. The presence of secondary metabolites indicates the importance of the two plants as therapeutic agents.

Table 1: Results of phytochemical studies.

The results of antimicrobial assay are given in table 2. BDME possessed no antibacterial or antifungal activity. In case of VEME, antibacterial activity was found against P. mirabilis with zone of inhibition of 9.0±0.2 mm and antifungal against S. cerevisae with zone of inhibition of 8.0±0.1 mm. The presence of antibacterial and antifungal activity in Vernonia elaeagnefolia indicates its usefulness as an effective antimicrobial agent against infectious clinical strains. The study can be further extended to standard strains to confirm the antimicrobial properties of the plant. The results of standard drugs Chloramphenicol (30µg) and Amphotericin (10µg) are also given in the table. 

The results of antioxidant assay are given in table 3. The percentage scavenging of BDME was found to be 95.35±0.00 whereas that of VEME was calculated to be 93.80±1.35. The antioxidant activity of BDME was equal to the standard drug BHT while VEME possessed slightly lower antioxidant and free radical scavenging activity than the two samples. The results show the presence of highly significant antioxidant activity in the two plants.

Table 3: Antioxidant activity of methanolic extract of B. disticha and V. elaeagnifolia.

DPPH assay is one of the easiest, quickest and most economical tests for the determination of antioxidant and free radical scavenging activity of plant extracts. Although this test alone is sufficient enough to establish the antioxidant value of plants however, some scientists perform additional tests to strengthen the results [10].


Natural products are one of the best sources of antioxidants. These antioxidants not only prevent our body from harmful free radicals but also prevent different food products and pharmaceuticals from getting spoiled. The present study proves the presence of high antioxidant activity in the two plants and therefore their beneficial role as therapeutic agents for the first time. Further studies may be carried out for detailed extract characterization and isolation of compounds responsible for the biological activity.


1.       Levy SB., Marshall B. (2004). Antibacterial resistance worldwide: causes, challenges and responses. Nature medicine, 10, 122-129.

2.       Walsh C. (2000). Molecular mechanisms that confer antibacterial drug resistance. Nature, 406(6797), 775-781.

3.       Amarowicz R., Pegg RB., Rahimi-Moghaddam P., Barl B., Weil JA. (2004). Free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Food Chemistry, 84(4), 551-562.

4.       De Boer HJ. (2012). Snake Gourds, Parasites and Mother Roasting: Medicinal plants, plant repellents, and Trichosanthes (Cucurbitaceae). Doctoral dissertation, Uppsala University, Sweden.

5.       Lorence DH., Flynn TW., Wagner WL., Evenhuis NL., Miller SE. (1995). Contributions to the flora of Hawai'i. III. New additions, range extensions, and rediscoveries of flowering plants. Bishop Museum Occasional Papers, 41, 19-58.

6.       Matthew KM. (1995). An excursion flora of central Tamilnadu, India. CRC Press, pp. 269-271.

7.       Raaman N. (2006). Phytochemical techniques. New India Publishing, pp. 5-36.

8.       Saad Touqeer., Muhammad Asad Saeed., Sharjeel Adnan., Farrukh Mehmood., Mueen Ahmad Ch. (2014). Antibacterial and antifungal activity of Melaleuca decora and Syngonium podophyllum. RJPT, 7 (7), 776-778.

9.       Singh RP., Chidambara Murthy KN., Jayaprakasha GK. (2002). Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. J Agr Food Chem, 50(1), 81-86. 

10.  Dudonne S., Vitrac X., Coutiere P., Woillez M., Mérillon JM. (2009). Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J Agr Food Chem, 57(5), 1768-1774.

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Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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