Faculty of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan.
Received: 21/07/2013 Revised: 18/08/2013 Accepted: 15/09/2013
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Mazus japonicus is an important plant of Scrophulariaceae family. In the present research work, the dichloromethane and methanol crude extracts of the plant Mazus japonicus were evaluated for phytotoxic, cytotoxic, antifungal and antibacterial activities. Both dichloromethane and methanol extracts of Mazus japonicus inhibited the growth of Lemna minor L. and showed significant phytotoxic activities. The dichloromethane extract showed cytotoxic activity at highest level of dose with LD50 513.04 μg/ml, while methanol extract showed no cytotoxic activity. The dichloromethane extract of the plant showed 25% inhibition against Fusarium solani and methanol extract of the plant showed 30% inhibition against Microsporum canis. Both dichloromethane and methanol extract of the plant exhibited non-significant activity when tested against pathogenic gram positive and gram negative bacterial strains. The phytochemical tests indicated the presence of saponins in the plant.
Mazus japonicus, Phytotoxicity, Cytotoxicity, LD50, Lemna minor L, Saponins.
Trees and plants are of paramount importance for human life, not only in the present time but also in the remote past as well. The early man depended on them for his physical needs such as source of food, shelter, clothing, medicines, ornaments, tools and for spiritual needs such as magic.
Herbs have always been used since prehistoric time till to date for curative purposes. An organized study of natural products, started at the beginning of 19th century, has been a pivotal factor in the development of potent biologically active molecules from plants. Therefore plants not only continued to retain their historical significance as one of the important source of new medicine for the treatment of diseases like cancer, acquired immunodeficiency syndrome (AIDS), malaria and disorders of cardiovascular and central nervous system, and many more. For all these reasons, bioactive molecules of plants have been always available to serve as source of inspiration to advance the traditional medicine and to prepare it to accept new challenges in time to come .
Majority of pharmacologically active molecules separated from plants are secondary metabolites, which are sophisticated arsenal to protect plants from outside dangers. Secondary metabolites perform important ecological functions including defenses against herbivores, bacterial and fungal infections. Many compounds used by the plants for defensive purposes can also be used by humans for the same purpose. However, secondary metabolites, which have ecological significance for plants, have altogether different effects on humans. For example, L-Dopa isolated from many leguminous plants like Vicia faba, acts as an antifeedant, but in humans it is used for the treatment of various psychatory disorders .
Plants have been used as medicines since beginning of human civilization. There are written evidences of medicinal uses of plants in texts of the ancient Chinese, Indian and other civilizations. India has had a history of ancient traditional medicinal practice based mostly on Ayurveda, Siddha and Unani systems of medicine. Medicinal plants have always been the main constituents of the traditional medicine. Ayurveda is based on natural products of nearly 2,000 cultivated and wild plant species. The written records of Ayurveda like Charaka Samhita, Shushruta Samhita and others contain more than 8,000 herbal remedies. There are literally millions of plants, combinations, traditions and household remedies to treat varieties of diseases and to boost health [3,4].
Scrophulariaceae is a large family with 220 genera and 3000 species distributed worldwide [5,6]. The genus Mazus of Scrophulariaceae family is eastern Asiatic, primarily of China. There are about 30 species in the genus, distributed in temperate and subtropical regions. In general the species of warmer regions and lower altitudes are widespread and of common occurrence. Some of the species of higher altitudes are restricted in their ranges. Among these species, the most widespread is Mazus japonicus, widely distributed in moist swampy places from India, China, Japan, south to the Philippines and Java, the only species that extends beyond the southern border of China [7,8,9].
The present research work was carried out in natural product chemistry laboratory and phytochemistry laboratory, Department of Pharmacy, Bahauddin Zakariya University, Multan, from January, 2012 to December, 2012. Brief description of materials as well as methods adopted is described below.
Collection and identification of plant
The plant material was collected in March, 2012 from surroundings of Bahauddin Zakariya University, Multan. The plant was identified as Mazus japonicus by Dr. Altaf Hussain Dasti, Professor, Institute of pure and applied Biology, Bahauddin Zakariya University, Multan and assigned a catalog no. STW 653.
Preparation of plant extracts
To achieve efficient extraction, the whole plant was dried under shade for 30days, then grinded it to a uniform powder and weighed. Simple maceration was adopted for effective extraction. Extract was prepared by soaking 200g of the dry powdered plant material of Mazus japonicus in a measured volume of dichloromethane in a closed container along with vigorous shaking for 24hrs and then filtered. This procedure was adopted three times with dichloromethane. The extraction of marc was carried out with methanol using same procedure. Rotary evaporator was used to concentrate the dichloromethane and methanol extracts. The extracts of dichloromethane and methanol were taken in different vials and named as MJD and MJM respectively.
Determination of Phytochemical Constituents
Phytochemical studies were carried out to analyze the presence of secondary metabolites. Alkaloids were detected by using Dragendorff, Mayer, Wagner and Hager’s Reagent. For detection of free and bound anthraquinone glycosides, Borntrager and modified Borntrager tests were performed. Keller Kiliani method was used for the confirmation of cardiac glycosides. Froth test was employed for the detection of saponins and for tannins Ferric chloride, Gelatin and Catechin tests were performed [6,10,11].
Lemna minor for Phytotoxicity Assay
Inorganic (E-medium) was prepared in which KOH pellets were added to attain pH 5.5-6.0. For testing, 10 vials per dose (500, 50, 5 ppm, control) were prepared. 15g of extract was dissolved in 15 ml of solvent. 1000, 100 and 10 μl solutions were dissolved to vials for 500, 40 and 5 ppm, after which solvents were allowed to evaporate overnight. 2 ml of E-medium and then a single plant containing healthy and green rosettes of three fronds were added to each vial. Vials were placed in a glass dish that is filled with 2cm of water. Then container was sealed. Dish with vials were placed in growth chamber for seven days at 26°C along with other controlled conditions. After three and seven days number of fronds per vial were counted and recorded. In the end, data was analyzed as percent of control with ED50 computer program to determine FI50 values and 65% confidence interval [12,13].
Brine-Shrimp Lethality Assay
The dichloromethane and methanol extracts of Mazus japonicus were tested for their cytotoxic activity by performing Brine shrimp (Artemia salina) lethality bioassay. For this assay mature shrimp larvae were produced by placing them in artificial sea water. For testing, three vials each of concentration 1000, 100 and 10 μg/ml were prepared. Then, 20mg of extract was added in 2ml of solvent. From these solutions 500, 50, or 5 μl were transferred to vials having 1000,100, or 10 μg/ml respectively. Solvent was allowed to evaporate. Then 5ml of sea water and 10shrimp larvae were added in each vial with the aid of Pasteur pipette. These vials were placed under illumination. After 24 hours surviving shrimps were counted and recorded and finney computer program was used to analyze data and to determine LC50 values and 95% confidence intervals [12,14,15].
Agar Tube Dilution Assay
The dichlorometane and methanol extracts of Mazus japonicus were tested against Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata. A control experiment with test substance was performed to evaluate the antifungal activity of extracts. Stock solution was prepared by adding dichlorometane and methanolic extracts in DMSO. Sabouraud Dextrose Agar (SDA) was prepared and a known volume was transferred into screw capped test tubes which were autoclaved at 121°C for 15 minutes and then test tubes were cooled to 50°C. The test samples were transferred from stock solution to the non-solidified Sabouraud agar media. Tubes were then solidified at room temperature. 4mm diameter of inoculums removed from a seven day old culture of fungi was inoculated in each tube. Inhibition of fungal growth was observed after 7-10 days of incubation at 28±1°C [12,15,16].
Agar diffusion method
The dichlorometane and methanol extracts of Mazus japonicus were tested against Bacillus subtilis (NCTC 8236), Staphylococcus aureus (NCTC 6571), Salmonella typhi, Escherichia coli (NCTC 10418), Shigella flexenari and Pseudomonas aeruginosa (ATCC 10145) by using this method. To determine antibacterial activity by using this method already prepared plates of broth culture media were used. From these plates required number of holes was cut by using a sterile cork borer keeping in view the proper distribution in the periphery and one in the centre. By using 0.1ml sterile pipette, 100μl of the dichloromethane and methanolic extracts in a suitable solvent, standard antimicrobial agent and solvent were poured into their respective hole. These plates were placed at room temperature for 2 hrs to get proper diffusion of the sample and incubated at 37°C for 24 hr. The diameter of the zones of inhibition was measured to the nearest mm [12,16,17].
Preliminary Phytochemical Tests
For the detection of secondary metabolites preliminary phytochemical tests were performed. The results of phytochemical tests showed the presence of saponins as a major class of secondary metabolites in Mazus japonicus. Results also indicated the absence of alkaloids, cardiac glycosides, anthraquinone glycosides and tannins in Mazus japonicus.
Saponins have been reported earlier to protect acute and chronic heart disease by exerting strong antioxidant activities by neutralizing reactive oxygen species. Cardiotonic effect showed by saponins was due to calcium channel blocking. Saponins are responsible for significant decrease in platelet aggregation. Saponin also showed protective effect on CNS. Saponins have been reported earlier to inhibit cancer cell proliferation indirectly through cell cycle protein, apoptosis related proteins, growth factors and protein kinases . Saponins also showed abortifacient, antizygotic and antiimplantation activity. Saponins were also found to be involved in enhancing immune system by cell mediated immune system and antibody production . Saponins are identified as a major class of secondary metabolites in the plant Mazus japonicus. It is conceivable that saponins hold a wide range of therapeutic potential and could be a lead for isolation and purification of individual saponins and can be put to further evaluation for their therapeutic potential.
The Lemna minor assay is a quick measure of phytotoxicity of plants extracts . The results of phytotoxic bioassay of crude dichloromethane and methanol extracts of Mazus japonicus are given in the Table No 2.
The results of phytotoxic bioassay showed that the dichloromethane extract inhibited the growth of Lemna minor L. upto 100% at high dose, 70% at moderate dose and 55% at low dose. While methanol extract inhibited the growth of Lemna minor L. upto 45% at high dose, 25% at moderate dose and 5% at low dose. Several workers reported the phytotoxic activity of various medicinal like Abroma augusta seed oil inhibited the growth of Lemna aequinoctialis . It has been reported that Chamaesyce hyssopifolia, Melaleuca quinquenervia, Acacia farnesiana, Ageratum conyzoides and Alphitonia excelsa showed phytotoxicity against Lemna aequinoctialis . Hussain et al. reported that n-hexane, n-butanol, chloroform and water fractions of Nepeta juncea showed insignificant phytotoxic effect against L. minor . The Lemna assay is a quick measure of phytotoxicity. The phytotoxicty bioassay is a useful primary screen for weedicide research. As weeds is one of the major factors of poor agricultural productivity in the developing countries. Synthetic weedicides are expensive, toxic and non specific. Weedicides from natural sources having improved characteristics could have a promising future [12, 24]. Phytotoxic natural products may be utilized either directly or as lead compounds for the development of herbicides. Furthermore, studies may be carried out to explore the phytotoxic components of the plant by isolation, purification and structure determination leading to the development of an effective herbicide.
Brine shrimp (Artemia salina) lethality bioassay was performed to evaluate cytotoxic activity of dichloromethane and methanol extracts of Mazus japonicus. Results of Brine shrimp (Artemia salina) lethality bioassay of crude dichloromethane and methanol extracts of Mazus japonicus are given in the Table No 3.
The dichloromethane extract showed cytotoxicity at highest level of dose with LD50 513.04 μg/ml, while methanol extract showed no cytotoxicity with LD50 2894.93 μg/ml. Brine-shrimp lethality bioassay has been used extensively to monitor the cytotoxicity of the sample under study. This is a rapid, inexpensive, in house, general bioassay which has been developed for screening, fractionation and monitoring of physiologically active natural products .
In vitro antifungal bioassay was performed to evaluate the antifungal activity of Mazus japonicus. In vitro antifungal bioassay was performed by following Agar tube dilution protocol. Results of In vitro antifungal bioassay of crude dichloromethane and methanol extracts of Mazus japonicus are given in the table 4.
The dichloromethane and methanol extracts of Mazus japonicus showed non-significant activity against Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani, Candida glabrata. It has been noted that dichloromethane extract showed 25% inhibition with linear growth at 75mm, when compared with control; only against Fusarium solani and methanol extract showed 30% inhibition with linear growth at 70mm, when compared with control; only against Microsporum canis.
The results of antifungal activity of the plant is in consistent with other plants of family Boraginaceae like Echium rauwolfii, Echium horridum , Cordia alliodora , Cordia linnaei , Cordia morelosana , Arnebia euchroma , Arnebia hispidissima , Trichodesma amplexicaule  and Cynoglossum officinale . Moderate activity was reported by crude methanolic extract of Onosma griffiithii against Aspergillus flavus (55%) and Fusarium solani (40%) while its n-butanol and ethyl acetate extracts shows no activity . No antifungal activity was recorded when aqueous and ethanolic extract of the plant, Colendia procumbens was tested against Candida albicans . Aqueous and methanol extracts of Anchusa italic and Trichodesma zeylanicum also displayed no activity .
In vitro antibacterial bioassay was performed to evaluate the antibacterial activity of Mazus japonicus. In vitro antibacterial bioassay was performed by using Agar tube diffusion method. Results of In vitro antibacterial bioassay of crude dichloromethane and methanol extracts of Mazus japonicus are given in the table 5.
The dichloromethane and methanol extracts of Mazus japonicus were tested against Eschericha coli, Bacillus subtilis, Shigella flexinari, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi by using Agar tube diffusion method. Both the extracts showed no activity.
The present findings have clearly demonstrated that the plant has excellent potential towards the phytotoxic and cytotoxic activity. The growth inhibition of the plant against Microsporum canis and Fusarium solani identified that the plant can be used as antifungal agent for the treatment of different fungal diseases. During preliminary phytochemical screening saponins are identified as a major class of secondary metabolites in this plant. So the further studies may be carried out to purify the active constituents of the plants that are responsible for its phytotoxic, cytotoxic and antifungal activity to get a valuable herbicidal, cytotoxic and antifungal agent. It is conceivable that saponins hold a wide range of therapeutic potentioal and could be a lead for isolation of therapeutically active novel saponins.