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Antimicrobial and Physiological Activities of different plant
parts of Rumex vesicarius L.
⃰ Nermien H. Seddek 1 & Marwa MR. M. Ahmed 2
1Botany & Microbiology Department, Faculty of Science, Assiut University, Egypt. ⃰
2Botany Department, Faculty of Science, Assiut University-New Valley Branch, Egypt.

⃰ Correspondence: or [email protected]

ABSTRACT

The objective of this study was to investigate the antimicrobial and physiological
(antioxidant capacity and total phenolic content) activities in order to find possible sources for antibacterial and antifungal drugs in addition to future novel antioxidants in food and pharmaceutical formulations from different plant parts of Rumex vesicarius L. as common wild medicinal plant. Leaves parts were the most active methanolic extracts against all tested positive and negative bacterial and fungal isolates. Also, highest antioxidant capacity and phenolic content were found in leaves extract. The high scavenging property of leaves may be due to hydroxyl groups existing in the phenolic compounds. This study demonstrated that Rumex vesicarius exhibited high antioxidant capacity for phosphomolybdate reduction. Obtained results showed a positive relationship between antioxidant capacity and total phenolic content along with the antimicrobial activity studies. The highest antioxidant capacity and total phenolic content recorded in leaves and this may be the main reason enabling leaves to give higher antimicrobial activity. Keywords: Rumex, Anibacterial, Antifungal, Antioxidant, Phenolic content
Native plants are an integral part of all healthy ecosystems. Each species contributes to a balanced system developed over millions of years, and interacts to keep the ecosystem functioning. Rumex vesicarius L. (Polygonaceae) is a wild edible plant used as a sorrel and collected in spring time and eaten fresh or cooked. R. vesicarius is effective against free radical mediated diseasesR. vesicarius L. has many important medicinal uses such as treatment of tumors, hepatic diseases, bad digestion, constipation, calcules, heart troubles, pains, diseases of the spleen, hiccough, flatulence, asthma, bronchitis, dyspepsia, piles, scabies, leucoderma, toothache and nausea. The plant also used as cooling, laxative, stomachic, tonic, analgesic, appetizer, diuretic, astringent, purgative, antispasmodic and antibacterial agents (Batanouny, 1999; Al‐Quran, 2009; Abutbul et al., 2005 and Lakshmi et al., 2009). A renewed interest has occurred in the last decade to search for phytochemical of native plants for antimicrobial purposes, many efforts have been done to found new antimicrobial compounds from various kinds of plants can possess antimicrobial natural products that can be used against infections (Cowan, 1999). Phytochemical screening of R. vesicarius L. found that all plant parts were rich in flavonoids, anthraquinones, alkaloids, tannins, sterols and/or triterpenodis, carbohydrates and/or glycosides, chlorides and sulphates and sublimable substances (Mostafa et al., 2011). Rumex vesicarius gave positive tests for alkaloids, anthraquinones, flavonoids, sterols (and/or terpenes) and tannins (Rizk et al., 1986). In the present scenario of emergence of multidrug resistance to human pathogenic infections, it has become very necessary to search for new antimicrobial substances from other sources (Doughari et al., 2007; Talreja, 2010; Khaled et al., 2010; Senthilkumar et al., 2011). Remarkable progress in the discovery of antimicrobial drugs has been made and thus several numbers of synthetic drugs have also been synthesized. These new synthetic antimicrobial drugs play a vital role in treatment of various types of microbial infections and reducing the number of fatalities that are associated with infections caused by microbes. It is also worth to note that in where medicines are quite expensive, investigation on antimicrobial activities frmay still be needed. Nonetheless, plants used in folk medicine in most developing are still understudied, particularly in clinical microbiology (Kirby, 1996; Wills et al., 2000; Pavrez et al., 2005; Zakaria et al., Material and Methods
Plant Material:
Rumex vesiacarius L. plant parts and soil samples were collected from Ain-Sokhna, Porto El-Sokhna resorts, Egypt. Plant specimens were botanically identified by comparing with herbarium specimens in the plant herbarium of Botany and Microbiology Department, Faculty of science, Assiut University, Assiut, Egypt. Preparation of Plant Extract:-
Leaves or aerial parts or flowers of the plants were collected randomly from Porto El
Sokhna Resorts, dried, and grinded by mechanical mixer to become powder, 100 g of each powder left in methanol for two days on mechanical shaker before filtration. This process was repeated three times to extract most of internal substance of the plant. The combined methanol extracts were transferred into rotatory evaporator under reduced pressure till semisolid residue was obtained. Then dissolved with Determination of Antimicrobial activity of extracts:
The disc diffusion method (Sleigh and Timburg, 1981) was used to measure the
antimicrobial activities of the tested plant extract, 0.1 g of each plant part extract dissolved in 2.5 ml dimethyl sulfoxide (DMSO) to give 4% dilution. Tested Microorganisms:
Bacteria: Eleven isolates of bacteria were tested as following : five isolates of Gram
positive bacteria namely: Bacillus subtilis (Ehrenberg) Cohn , B. cereus (Frankland & Frankland), Enterococcus faecalis Schleifer & Kilpper-Bälz, Micrococcus leuteus (Schroeter) Chon and Staphyllococcus aureus (Rosenbach), and six isolates of Gram negative bacteria namely: Escherichia coli (Migula) Castellani & Chalmers, Klebsiella pneumoniae (Schroeter) Trevisan, Proteus vulgaris Hauser, Pseudomonas aeroginosa (Schroeter) Migula, Salmonella typhi (Schroeter) Warren & Scott and Serratia marcenscens Bizio. All bacterial isolates were kindly obtained from Department, of Clinical Pathology Faculty of Medicine, Assiut University. Toxinogenic Fungi: Three isolates of Toxinogenic fungi namely: Aspergillus flavus,
A. fumigatus and F. moniliforme were obtained from laboratory of fungi, Botany Department, Faculty of Science, Assuit University. Medical Fungi: Four isolates of the dermatophytic fungi namely: Trichophyton
rubrum (castell) Semon, T. mentagrophytes (Robin) Blanchard, Sabouraudites canis (E. Band Epidermophyton floccosum Langeron & Miloch were used to determine the anti-dermatophytic activity of the tested plant extract. These isolates obtained from Assiut University Hospital isolated from patients affected by onchomycosis, tinea capitis and tinea corporis. Three isolates of pathogenic Candida species were obtained from the AUMC. They were previously isolated from women complaining of vaginal candidiasis (Farhan, 2011). These isolates were Candida
albicans (Robin) Berkhout, Candida glabrata (Anderson) S.A. Meyei & Berkhout and Candida krusei (Castellani) Berkhout. Used Media:
Nutrient agar (Collee et al, 1996): used for growing of the bacterial isolates; it
composed of Beef extract, 10 g; peptone, 10 g; NaCl, 5 g; agar, 15 g (g/L) and
adjusted to pH=7 before sterilization at 121ºC for 20 minutes.
Czapek's agar (Thom and Raper, 1945): used for growing the toxigenic fungal
isolates; it composed of glucose, 10 g; NaNO3, 3g; K2HPO4,1 g; Mg SO4, 0.5 g;
KCl, 0.5 g; and agar 15 g (g/L) and adjusted to pH=6.5 before sterilization at 121ºC
for 20 minutes.
Sabaroud dextrose agar (modified by Emmons): used for growing the medical
fungal isolates; it composed of glucose, 20 g; peptone, 10 g; and agar, 15 g/L and
adjusted to pH=5.5 before sterilization at 121ºC for 20 minutes.
Analysis of Some Soil Samples:
Soil PH : Soil PH determined by PH meter (Jackson, 1958).
Determination of Total Soluble Salts: known weight of each tested soil sample was
shaken in a known volume of distilled water for about 30 minutes by mechanical
shaker. Then the mixture was left overnight to precipitate. The soil extract was
filtered and a known volume was transferred into a weighted crucible and was then
evaporated in an oven at 105 C . The dry residue was weighed and the total soluble salts percentage was calculated. Water Content of Soil: determined by drying 10 gm of soil sample overnight at hot
air over 105 ºC till reached to constant weight. The loss of weight was determined,
and then the water content was calculated. Percentage of moisture content was
calculated by the following equation: [% moisture content = A-A1/A] Where, A=
weight of soil before drying and A1= weight of soil after drying.
Determination of Organic Matter: by Welkely-Black method (Jackson 1958). A
known amount of sieved soil was digested by chromic acid (for oxidation of organic
matter to Co2) and the excess amount of chromic acid was back titrated against
standard ferrous sulphate solution in presence of 2 drops of diphenylamine as an
indicator.
Determination of Cations:
1- Calcium and Magnesium (Ca++ and Mg++) were determined by versene (Disodium Dihydrogen Ethylene Diamine Tetra Acetic Acid) titration method which described by Schwarzenbah and Biedermann (1948). 2- Sodium and Potassium (Na+ and K+): Flame emission technique is the most rapid and sensitive method (Williams and Twine, 1960). Carl Zeiss flame photometer was used. Determination of Chlorides: It is performed according to the silver nitrate titration
method using potassium chromate as an indicator (Jackson 1958).
Estimation of Total Phenolic Content:
The total phenolic content was determined spectrophotometrically in methanol plant
extract (Kim et al., 2003). 1 ml of sample (1 mg/ml) was mixed with 1 ml of Folin-
Ciocalteu's phenol reagent. After 5 min, 10 ml of a 7% Na2CO3 solution was added to
the mixture followed by the addition of 13 ml of de-ionized distilled water and mixed
thoroughly. The mixture was kept in the dark for 90 min at 23°C, after which the
absorbance was read at 750 nm. The TPC was determined from extrapolation of
calibration curve which was made by preparing gallic acid solution. The estimation of
the phenolic compounds was carried out in triplicate. The TPC was expressed as
milligrams of gallic acid equivalents (GAE) /g of dried sample.
Phosphomolybdate Assay [Total Antioxidant Capacity]:
The total antioxidant capacity in methanol plant extract was determined by
phosphomolybdate method using ascorbic acid as a standard (Umamaheswari and
Chatterjee, 2008). An aliquot of 0.1 ml of sample solution was mixed with 1 ml of
reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate). The tubes were capped and incubated in a water bath at 95°C
for 90 min. After the samples had cooled to room temperature, the absorbance of the
mixture was measured at 695 nm against a blank. A typical blank contained 1 ml of
the reagent solution and the appropriate volume of the solvent and incubated under
the same conditions. The antioxidant capacity was expressed as Gallic Acid
Equivalent (GAE) by using the standard Gallic acid graph.
Statistical Analysis:

The standard deviation analysis (SD) was employed for statistical analysis of the plant extracts antimicrobial activities results. Results:
Antimicrobial activities of different plant parts methanol extract
The study reported here deals with the inhibitory effects of the methanolic extracts of the studied different parts plant of Rumex vesicarius L. on the growth of various isolates (tables 1 , 2). 100µg of each tested methanolic plant part extract was added to sterilized filter paper disc (Whatman No.3, 0.5 cm diameter) and then placed on to the lawn of microorganisms. Standard antibiotic (Gentamycin= Epigent ampoules 2ml-Epico company) was co-assayed as positive control while sterilized filter paper saturated with 100µg DMSO used as a negative control. Table (1): Antibacterial activities of methanolic extracts of different plant parts
against different pathogenic bacterial strains
Table (2): Antifungal activities of methanolic extracts of different plant parts against
different fungal isolates
Inhibition zone (mm) ± standard Bacillus subtilis Bacillus cereus Enterococcus faecalis Micrococcus Staphylococcus Escherichia coli Klebsiella pneumonia Proteus vulgaris Pseudomonas aeruginosa Salmonella All results recorded as mean values of three replicates. Potency assessed by measuring inhibition zones (mm) ± standard deviation in relation to those of positive and negative controls. These results revealed that, different plant parts methanolic extract displayed a broad spectrum and variable degree antimicrobial activity against Inhibition zone (mm) ± standard isolates Aspergillus A.fumigatus moniliforme Candida albicans Candida Candida krusei Trichophyton rubrum T. mentagrophytes Sabouraudites canis Epidermophyton floccosum tested strains depend on kind, strength and resistance of the tested microorganism. No inhibition activity observed with DMSO against any strain of the all bacterial and fungal isolates. Among all methanolic R. vesicarius leaves and stems extracts demonstrated high antibacterial activities against all tested Gram positive and negative bacterial strains with exception of Klebsiella pneumonia was resistant against stems extracts. The flower methanolic extracts had inhibitory effects against all tested Gram positive bacterial strains while all tested Gram negative bacterial strains were resistant except Pseudomonas aeruginosa (8 ±0.2). Gentamycin as standard antibiotic demonstrated effective inhibitory effect against all tested bacterial strains. The magnitude of anti-fungal dermatophytic and anti-candida activities were assessed by the diameter of inhibition zones relative to those of positive and negative controls, standard antifungal (Fluconazole discs obtained from HiMedia Company, India) was co-assayed as positive control. From Data in table 2 it is evident that methanolic extract of leaves showed anti-dermatophytic and anti toxinogenic fungal activities against all tested pathogenic fungal dermatophytes and toxinogenic fungal isolates with range 5±0.1 to 13±0.2 while it had no activity against tested Candida strains except C.albicans (8 ±0.3). It is worth to be noted that Candida albicans was more sensitive than C. glabrata and C. krusei to all methanolic extracts of studied different Methanol extract of stems displayed inhibitory effect against all tested fungal isolates recorded in table 2 except Trichophyton rubrum, C. glabrata and C. krusei while methanol extract of flowers displayed no inhibition activities against all tested fungal isolates except A.fumigatus and Candida albicans. Standard antifungal disc of Fluconazole had inhibitory effects against all tested fungal isolates with inhibition zone range 2 ±0.1 against Candida glabrata to 18 ±0 against Epidermophyton floccosum. Analysis of Soil Samples:
Data recorded in (table 3 and 4) determined the soil analysis of some ten soil samples from the same location of the tested plant, this results revealed that tested soil samples exhibits alkaline PH (more than 7). No obvious extremes in PH values among the different tested samples, therefore the range of PH was from 7.2 to 8.1. Percentage of total soluble salts (TSS %) highest value was 2.22 and lowest value was 1.24 at the soil sample no.4 and 9 respectively. Variation of the organic matter content (OMC %) among the tested samples was not obvious, highest value was 0.52 and lowest value was 0.23 at the soil sample no.4 and 8 respectively. Data of soil moisture content (MC %) nearly the same at the different tested samples, it was nearly about 0.3. From data shown in table 4, there were no obvious differences among the ten collected soil samples. Analysis of soil samples recorded highest amounts of Ca+2, Mg+2, Na+, K+ and Cl- cations as follows ( 0.082, 0.041, 0. 480, 0.072 and 0.71) mg/g dry soil. Table (3): Percentage of total soluble salts (TSS %), organic matter content (OMC %)
and moisture content (MC %) and PH values of the ten studied soil samples.
Table (4): Calcium, Magnesium, Sodium, Potassium and Chloride elements (mg/g dry
soil) of the ten studied soil samples:
Antioxidant Activity and Total Phenolic Content:
Data recorded in table 5 showed the antioxidant activities and total phenolic content
of different plant parts which estimates in this investigation. There were observed
variations in the antioxidant activities and total phenolic content within the different
tested plant parts. The higher Antioxidant activity and total phenolic content is
recorded in leaves methanol extract (it was 29.82±0.3 ppm and 15.12±0.33 mg GAE/g
FW respectively). Flowers showed the lowest antioxidant capacity and total phenolic
content (it was11.61±0.2 ppm and 9.123±0.52 mg GAE/g FW respectively).
Table (5): Antioxidant Activity (Gallic acid equivalent "ppm") and Total Phenolics (mg
GAEs/g FW) of different plant parts of Rumex vesicarius L.
Plant parts
Total Antioxidant Activity
Total Phenolics
29.82 ±0.3
15.12 ±0.33
26.63 ±0.5
14.46 ±0.12
11.61 ±0.2
9.123 ±0.52
Discussion
Methanol extract of different plant parts of R. vesicarius displayed a broad spectrum and variable degree antimicrobial activity against tested strains depend on kind, strength and resistance of the tested microorganism. Methanol extract of R. vesicarius leaves and stems demonstrated high antibacterial activities against all tested Gram positive and negative bacterial strains with exception of Klebsiella pneumonia. These
results agree with Mostafa et al., 2011, they evaluated the antibacterial activities of successive extractives solvents (petroleum ether, ether, chloroform, methanol and ethanol) of different plant parts of R. vesicarius. Their results revealed that, there were highly significant variations within antibacterial activities of different extracts of different plant parts and methanol extract of roots was found to be the most effective one against Streptococcus pneumoniae (inhibition zone= 18.000 mm, activity index= 0.328) and ethanol extract of flowers was found to be the most effective one against Escherichia coli (inhibition zone = 15.875 mm, activity index= 0.508). Also, results of antibacterial activities were in harmony with results of Panduraju et al., 2009 who found that, aqueous, methanol and petroleum ether extracts of Rumex vesicarius L. leaves have variable effects against both gram‐positive and gram‐negative bacteria.The flower methanolic extracts had inhibitory effects against all tested Gram positive bacterial strains while all tested Gram negative bacterial strains were resistant except Pseudomonas aeruginosa (8±0.2). It appears that Gram negative bacteria are resistant to many natural products including essential oils and other metabolites extracted from medicinal plants. The reason for differential sensitivity between Gram-positive and Gram-negative bacteria could be ascribed to the morphological differences between these microorganisms; Gram-negative bacteria have an outer polysaccharide membrane carrying the structural lipopolysaccharide components. This makes the cell wall impermeable to lipophilic compounds; on the other hand, Gram +ve bacteria will be more susceptible as they have only an outer peptidoglycan layer which is not an effective permeability barrier (Gao et al., 1999). Extract of leaves was the most active one against all tested fungal isolates, it showed anti-dermatophytic and anti toxinogenic fungal activities against all tested pathogenic fungal dermatophytes and toxinogenic fungal isolates with range 5 ±0.1 to 13 ±0.2 while it had no activity against tested Candida strains except C.albicans (8 ±0.3), so this revealed that R. vesicarius had antifungal potency. In our knowledge, there is no previous literatures in this way. Several techniques have been used to determine the antioxidant activity in vitro in order to allow rapid screening of substances since substances that have low antioxidant activity in vitro, will probably show little activity in vivo (Nunes et al., 2012). Antioxidants fight against free radicals and protect us from various diseases. They exert their action either by scavenging the reactive oxygen species or protecting the antioxidant defense mechanisms (Umamaheswari and Chatterjee, 2008). The antioxidant capacity of the methanolic extracts of different plant parts were measured spectrophotometrically through phosphomolybdenum method, based on the reduction of Mo (VI) to Mo (V) by the test sample and the subsequent formation of green phosphate/Mo (V) compounds with a maximum absorption at 765 nm. The antioxidant capacity of the methanolic extracts of different plant parts ranging from 11.61 ± 0.2 in flowers to 29.82 ± 0.3 in leaves. Soil structure affects plant growth in many ways. Due to the great importance of R. vesicarius L. plant and in order to know more about the soil in which our plant grows, some soil analysis is registered. Average PH of these soil samples is about 7.72 (alkaline soil). Analysis of soil samples recorded average amounts of Ca+2, Mg+2, Na+, K+ and Cl- cations as follows ( 0.066, 0.026, 0.342, 0.062 and 0.61) mg/g dry soil. Percentage of total soluble salts (TSS %) reported around 1.7 at the different soil samples tested. Variation of the organic matter content (OMC %) among the tested samples was not obvious. The average amount was about 0.0042. Data of soil moisture content (MC %) nearly the same at the different tested samples, it was nearly about 0.3. These measurements revealed the suitable soil components for the cultivation and growth of R. vesicarius L. plant. Large number of medicinal plants has been investigated for their antioxidant properties. Natural antioxidants either in the form of raw extracts or their chemical constituents are very effective to prevent the destructive processes caused by oxidative stress (Saeed et al., 2012). Antioxidants are crucial in the prevention of human diseases. Because of its high antioxidant activity, R. vesicarius is effective against free radical mediated diseases. In this study methanol extract only used according to (Saeed et al., 2012) who found that the methanol extract exhibited the highest total phenolics content, whereas the contents obtained with residual aqueous fraction were much smaller that is in agreement with other reports (Ao et al., 2008). The present antioxidant activity results of R. vesicarius L. were approximately lesser than Nishina et al., 1991; Demirezer et al., 2001; Al-Ismail et al., 2006; Özen, 2010 and Li and Liu, 2009 since they investigated different extracts of roots of R. japonicus and R. patientia, leaves of R. pulcher and R. acetosella and whole plant parts of R. dentatus respectively and they proved that these species were considered to be antioxidant agents. The present work demonstrated that the different plant parts (leaves, stems and flowers) of R. vesicarius exhibited high antioxidant capacity for phosphomolybdate reduction. Total phenolic content of the different plant parts expressed as milligrams of gallic acid equivalents (GAE) equivalent; it ranged from 9.123 ±0.52 in flowers to 15.12 ±0.33 in leaves. Generally, plant organs which rich in phenolic content are increasingly being used in the food industry because they retard oxidative degradation of lipids and improve the nutritional value and quality of the food (Kähkönen et al., 1999). Phenolic compounds are considered secondary metabolites and these phytochemical compounds derived from phenylalanine and tyrosine occur ubiquitously in plants and are diversified (Naczk and Shahidi, 2004). Herbal compounds with antioxidants activity may function as free radical scavengers, reducing agents and quenchers of single oxygen formation or reactive oxygen species, thereby protecting the body from degenerative disease such as cancer. The reactive oxygen species are damaging byproducts generated during normal cellular metabolism or from toxic insult. They lead to a state of oxidative stress that contributes to the pathogenesis of a number of human diseases by damaging lipids, proteins and DNA (Khan et al., 2014). Antioxidant activity and total phenolic content results agreed with El-Hawary et al., 2012, where R. vesicarius had antioxidant and hepato-protective effects due to the presence of phenolics and flavonoids also with results of Tavares et al., 2010, since they found that, flavonoids and polyphenolics in R. maderensis were strongly associated with antioxidant capacity, total flavonoids and phenolics content reflecting the antioxidant capacity of the plant. Phytochemical screening of R. vesicarius L. found that all plant parts were rich in flavonoids, anthraquinones, alkaloids, tannins, sterols and/or triterpenodis, carbohydrates and/or glycosides, chlorides and sulphates and sublimable substances (Mostafa et al., 2011). This may be the reason for the highly antimicrobial activities obtained by these extracts. These results were in harmony with results of Park et al., 2006; Cushnie and Lamb, 2005; Matkowski, 2008 and Rao, 2003 whereas they found that, flavonoids and polyphenols were good antioxidant and antibacterial gents, since Rumex vesicarius L. is rich in polyphenols, so it can be found that, there were a positive relationship between chemical composition and the obtained biological activity results of this plant, so the presence or absence of flavonoids may be a determining factor of this biological activity. It can be concluded that Egyptian medicinal plant R. vesicarius L are excellent source for bioactive therapeutic compounds used as anti- bacterial, anti- fungal and antioxidant agents, therefore it can be used for the drugs References
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