Antioxidant , Antimicrobial and Antidiarrhoeal Activity of Methanolic Extract of Rumex maritimus L . ( Polygonaceae )

Article history: Received on: 15/07/2015 Revised on: 14/08/2015 Accepted on: 01/09/2015 Available online: 12/11/2015 The aim of this current study was to evaluate phytochemistry along with pharmacological activities of the whole plant of Rumex maritimus L. (Family: Polygonaceae). Phytochemical analysis of the extract of R. maritimus indicates the presence of Reducing Sugar, Glycoside, Gum, Tannin and Alkaloid type compounds and the pharmacological attention of these compounds prompted us to check R. maritimus for possible anti-oxidant, antimicrobial and antidiarrheal activities in a dose dependent manner. The dried plants were subjected to successive extraction with methanol and the extract was used to investigate the activities. The extract of plants produced good diarrheal inhibition in castor oil induced diarrhea in mice (250mg/kg and 500mg/kg body weight) which was comparable to the standard drug Loperamide at the dose of 3 mg/kg of body weight. The alcoholic extract was showed anti-bacterial activity against the tested microorganisms (both gram positive as well as negative bacteria). This study reveals the potent anti-oxidant activity that is (IC50~80 μg/ml) to that of standard drug ascorbic acid (IC50 about ~7 μg/ml) in-vitro when tested in 1, 1-Diphenyl-2picrylhydrazyl (DPPH) scavenging method. The obtained results provide a support for the use of this plant in traditional medicine and its further investigation.


INTRODUCTION
Bioactive Guided Approach-Once the plant medication was provided to the ancient people in the crude form often exhibited many unwanted effects due to the presence of some toxic components beyond the active constituent.Wide-ranging phytochemical evaluation and isolation of active component(s) in the pure form thus become necessary to avoid untoward effects and to ensure safe use of herbal medicines.With technological advancement, phytochemical studies of medicinal plants got a rapid pace and the presence of many chemical compounds comes in light.These plant-derived compounds often played an important role in directing laboratory synthesis of many new classes of drug molecules.Sometimes the plant components became the starting material in the synthetic process of industrial production of many drug molecules like the application of .

Plant collection and Identification
For this present investigation the plant Rumex maritimus L. was collected from road side area of Nirala residential area Khulna City, Khulna, Bangladesh in February 2010 and was identified by Bangladesh National Herbarium, Mirpur, Dhaka.(Accession number-29865) and a voucher specimen was deposited in Khulna University laboratory.

Drying and grinding
Unexpected materials were separated from the collected medicinal plants and these were dried by shade drying for seventy days to ensure the active constituents free from decomposition as well as to avoid any photochemical degradation.The whole plants were ground into a coarse powder with the help of an appropriate grinder.In an airtight container, grinded powder was stored and kept in a cool, dark and dry place for analysis until commence.

Cold extraction (Methanol extraction) & Evaporation
The plants were extracted by cold extraction method.100 gm grinded powder was in 500 ml of methanol in a glass container for eight days accompanying regular shaking and stirring.Plant debris was separated from the extract by filtration by a piece of clean, white cotton material & it was done two times.
The filtrate (methanol extract) obtained was taken into rotary evaporator to evaporate methanol.
Then this filtrate was taken into beaker, the opening of beaker was wrapped by a sheet of aluminum foil to which perforation was done for evaporation of the rest of the methanol & was kept in dry & cool place for several days & at last evaporation was done under table fan until dried.It rendered concentrate of deep purple type and it was designated as crude extract of methanol.

Phytochemical Tests
Testing of different chemical groups present in extract, represent the preliminary photochemical studies-The chemical group tests, which are performed according to Evans (1989).In each test 5 % (w/v) solution of extract in methanol was taken unless otherwise mentioned in individual test.

Test for antimicrobial activity
To inhibit the development of microorganisms, some researchers use the diameter of zone of inhibition and/or the minimum weight of extract.Though, a great amount of factors like the extraction methods, inoculum volume, and culture medium composition (Bauer et al., 1966) pH and incubation temperature can stimulate outcomes.Among the above revealed techniques the disc diffusion (Bauer et al., 1966) is a widely accepted in vitro investigation for preliminary screening of test agents which may possess antimicrobial activity.On the other hand, no difference between bacteriostatic and bactericidal activity can be demonstrated by this method (Roland, 1982).
The total experiments were carried out three times and the mean of the reading were recorded.Microorganisms used for the activity test both gram positive and gram-negative bacterial strains were taken.The bacterial strains used for the investigation are listed in Table 1.These organisms were collected from the Microbiology Laboratory of Pharmacy Discipline, Khulna University, Bangladesh.

Methodology
Preparation of media-Nutrient agar media was prepared by adding water to a dehydrated product that contains all the ingredients.Practically all media are available commercially in powdered form (Pelczar, 1986).

Anti Oxidant test
For assessing the DPPH radical scavenging activity, the modified method described by Gupta et al., (2007) was used.
Stock solution (5mg/ml) of R. maritimus were prepared in and serial dilutions were carried out to obtain concentrations of 1, 5, 10, 25, 50,100, 200, 400 µg/mL.In this assay, an equal amount sample solution was added to an equal amount of 0.1mM methanolic DPPH solution, to occur.After about half an hour incubation period, the absorbance was took against a blank at 517 nm with a double beam Analykjena UV/visible spectrophotometer (Specord 205, Germany).Substances which perform this reaction can be considered as antioxidants and accordingly radical scavengers (Dehpour, 2009).The radical scavenging activity was expressed as the inhibition percentage (I%) and calculated as per the equation: Where A blank is the absorbance of the control reaction (containing all reagents except the test compound) and A sample is the absorbance of the test compound.IC so value is the concentration of sample required to scavenge 50% DPPH free .radical and was calculated from the plot of inhibition (%) against extract concentration.All the tests were done in triplicate and average of the absorptions was noted.Ascorbic acid was used as positive control standard.Ascorbic acid was used at standard compound to study the DPPH radical scavenging activity test.Ascorbic acid or Vitamin C is the most powerful anti-oxidant.Ascorbic acid was found active during this study while tested for DPPH radical scavenging.The percent hang-up of DPPH radical by the extracts was compared to a synthetic antioxidant i.e.Butylated Hydroxyanisole (BHA).All the fractions showed less radical scavenging activity that was compared with BHA.The activity of the extract which showed the highest DPPH radical scavenging activity (ethyl acetate) was about half of BHA at 100 ppm concentration.At about 200 ppm, the ethyl acetate fraction shows nearly 93% DPPH scavenging activity (data not given).Even though, when compared to BHA, the concentration required for an equivalent activity was higher for this fraction, there was wide spread settlement that BHA and butylated hydroxytoluene (BHT) to be exchanged with natural antioxidants considering their potential health hazards and harmfulness (Kahl and Kappus, 1993).

Anti-diarrheal Activity Screening
In pathological term, diarrhoea occurs due to passage of excess of water in faeces.In the ileum and colon active Na + K + ATPase mediated salt absorption occurs, water follows isoosmotically.In addition glucose facilitated Na + absorption take place in the ileum.This mechanism remains intact even in severe diarrhoeas.Diarrhoea associated with carcinoma (secreting 5-HT) and medullary carcinoma of thyroid (secreting calcitonon).It is mediated by cAMP.Excess of bile acids also causes diarrhea by activating adenylcyclase (Tripathi, 2001).Diarrhoea was defined by the presence of stool or any fluid material that stained the absorbent paper placed beneath the cage.Time taken before the first defecation is the 'Latent period'.The total count stool and latent period of test group are compared with positive control group.Anti-diarrhoeal agent increase latent period and decrease total number of stool.The effect of R. maritimus on castor oil (0.5 ml each mouse at 6 hr.) induced diarrhoea in mice in relative to standard, when we observed the t-value (P-value) along with Standard Deviation (SD) with Standard Error (SE).In table 5, shows the statistic analysis of effect of R. maritimus on castor oil (0.5 ml each mouse at 6 hr.) induced diarrhoea in mice.

For Phytochemical analysis
The crude extract was tested for the presence of different chemical groups and the following groups were identified-glycosides, tannins, gums, alkaloids, high level of reducing sugar etc.

For Anti-microbial activity
The crude extract of R. maritimus was tested for antimicrobial activity against a number (07) of both gram positive and gram-negative bacteria.A standard antibiotic disc of Kanamycin was used for comparison purpose.The crude extract of 500µg/disc showed anti-microbial activity against Streptococcus agatectiae, Shigella dysentrae and Staphylococcus saprophyticus (Table 1).Anti-microbial activity observed not only for gram positive but gram negative bacteria also.Other family member's of Polygonaceae, like R. dentatus, R. nepalensis, R. alveollatus, R. obtusifolius have their anti-bacterial activity, to judge these evidence we use whole plant of R. maritimes and finally got antimicrobial action.It is a preliminary investigation and further study should be done for more scientific evidence.

For Antioxidant activity
The methanolic extract of R. maritimus showed comparable antioxidant activity (IC 50 ~80 µg/ml) against DPPH assay (M. S. Blois, 1958) to that of standard drug ascorbic acid (IC 50 ~7 µg/ml).In figure 2, DPPH radical scavenging test of crude extract of R. maritimus and Figure 3, DPPH radical scavenging test of Ascorbic acid (Standard).The free radicals are created in aerobic cells due to ingestion of oxygen in cell progress (Rice- Evans, 2004, Morton, 1992).Free radicals cause reduction in membrane fluidity, loss of enzyme receptor action and impairment to membrane protein prominent for death (F. Franca, 1996).These free radicals are involved in various sicknesses like ageing, cancer, CVS diseases, diabetes, arthritis, epilepsy & ruin of essential fatty acids.Antioxidant helps in treatment of above disorders (Jain and Lata, 1996).In this study, percentage inhibition of free radical scavenging activity was increased with the increase concentrations of both crude extract and ascorbic acid.It showed maximum inhibition 85.71% at 500µg/ml and 50% inhibition at concentration near than 7µg/ml (figure 4).
From the above test and analytical method, it can be concluded that, the crude extract of R. maritimus has antioxidant activity.The free radical scavenging property may be one of the mechanism by which the plants are effective in traditional medicine.Further study is necessary to identify the constituents which are responsible for this property as well as studies with other models, such as lipid per-oxidation and in-vitro assays are essential to characterize them as biological anti-oxidants.

For Antidiarrhoeal activity
In the castor oil-induced diarrheal mice, the methanolic extract of R. maritimus at the doses of 250 mg/kg and gave large number of stools of stools along with less delay in first defecation i.e. shorter latent period comparing with standard drug, Loperamide.In figure 5 shows, the latent period of R. maritimus extract on castor oil induce diarrhoea in mice & in figure 6 shows: the effect of R. maritimus extract on the basis of mean stool count on castor oil induce diarrhea in mice.The ricinoleic acid creates irritating and inflammatory actions on the intestine which leads to the discharge of prostaglandins (Yoshio et al., 1999) and exciting peristaltic (decreasing Na+ and K+ absorption) activity and diarrhoea (Zavala et al., 1998).Loperamide is an opiate/alkaloid analogue (Tripathi, 2008) which inhibits prostaglandin synthesis and interruption diarrhoea induced with castor oil (Sunil et al., 2001).The presence of alkaloids of R. maritimus may have same pathway as Loperamide does.Furthermore, antidysenteric and antidiarrhoeal belongings of medicinal plants were found due to alkaloids as well as tannins, saponins, flavonoids, sterols and/or triterpenes and reducing sugars (Havagiray et al.,. 2004).These ingredients are also present in R. maritimus responsible for antidiarrhoeal activity.So it can be declared that it has a little antidiarrhoeal activity though the activity increased in a dose dependent manner.

Fig. 4 :
Fig. 4: Percentage inhibition of crude extract of R. maritimus and with ascorbic acid (Standard).

Fig. 5 :
Fig. 5: The latent period of R. maritimus extract on castor oil induce diarrhoea in mice.

Fig. 6 :
Fig. 6: The effect of R. maritimus extract on the basis of mean stool count on castor oil induce diarrhea in mice.

Table 1 :
List of gram positive and Gram negative strains.

Table 2 :
Statistic analysis of effect of R. maritimus on castor oil (0.5 ml each mouse at 6 hr.) induced diarrhoea in mice.