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Chemical and biological evaluation of Ranunculus muricatus

Article  in  Pakistan journal of pharmaceutical sciences · November 2015

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Chemical and biological evaluation of Ranunculus muricatus

Farhat Ali Khan1,2, Muhammad Zahoor2* and Ezzat Khan2

1
Department of Pharmacy, Sarhad University of Science and Information Technology, Peshawar, KPK, Pakistan
2
Department of Chemistry, University of Malakand, Chakdara Dir (Lower), KPK, Pakistan

Abstract: Ranunculus muricatus is commonly known as spiny fruit buttercup and is used in the treatment of intermittent
fevers, gout and asthma. Qualitative analysis of phytochemicals of Ranunculus muricatus indicated the presence of
saponins, tannins, phenols, flavonoids and alkaloids. Saponins were present in high amount as compared with other
chemicals. Inorganic and heavy metals constituents were determined. Heavy metals estimation in the sample showed that
iron was present in high amount followed by zinc even then the concentration of these metals is below acceptable limit.
The physical parameters, antioxidant and antimicrobial activities of the extracts were determined. Acetone extract
fraction showed optimal antioxidant activity as compared to ethanol and chloroform fractions of the candidate plant. The
antimicrobial and antifungal activities of the crude extract and extract fractions were determined by well agar diffusion
method. Highest zone of inhibitions were observed for crude extract followed by acetone extract fraction against
Micrococcus luteus. Antifungal activities were high for crude extracts against Candida Albican. Findings of this study
show that Ranunculus muricatus has a good medicinal impact.

Keywords: Ranunculus Muricatus; phytochemicals; inorganic constituents; antioxidants; antibacterial; antifungal.

INTRODUCTION scavenging reactive oxygen and nitrogen free radical

species, decreasing the localized oxygen concentration
Medicines resulting from plants are commonly famous thereby reducing molecular oxygen’s oxidation potential,
due to their protection, easy accessibility and low cost. metabolizing lipid peroxides to non-radical products and
Plant based products may contain whole parts of the plant chelating metal ions to prevents the generation of free
or mostly prepared from leaves, roots, bark, seed and radicals (Robinson and Maxwell, 1997).
flowers. They are administered orally, inhaled or directly
applied to the skin (Westh et al., 2004). The uses of plants Heavy metals in plants are the main constituents of the
as medicinal agents are due to the presence of important inorganic contamination. Heavy metal toxicity can result
phytochemicals like alkaloid, flavonoid, tannin, saponin in damaged or reduced mental and central nervous
and phenolic compound, which provides the basis of function, lower energy levels and damage to blood
modern drugs known today (Crag and Newman, 2001; composition, lungs, kidneys, liver and other vital organs.
Krishnaiah, 2007) Long-term exposure may result in slowly progressing
physical, muscular and neurological degenerative
Nowadays, multiple drug resistance has been developed processes that mimic Alzheimer's disease, Parkinson's
due to the indiscriminate use of commercial antibacterial disease, muscular dystrophy, and multiple sclerosis.
and antifungal agents used in the treatment of infectious Allergies are rare and occur through repeated long-term
diseases. These problems enforced scientists to investigate contact with some metals or their compounds that may
for new antimicrobial substances. Therefore, it is a needed even cause cancer (Fletcher, 1991).
to develop alternative antimicrobial drugs for the
treatment of infectious diseases from medicinal plants Health depends upon the organized state of elements like
(Lee, 2003). Antimicrobial agents of plant origin have Na, K, Ca, Mg, Cl, F etc in the body and their imbalance
enormous therapeutic potential. They are effective in the causes diseases. The restoration of balance by drug can
treatment of infectious diseases while simultaneously cure diseases (Farhat et al., 2011). The remarkable
mitigating many of the side effects that are often progress that has been made in the science of Medical
associated with synthetic antimicrobials (Byika et al., Elementology during the past few decades has not only
2003). opened avenues for research on human health related
aspects but also aroused the interest of the pharmaceutical
Anti-oxidants are chemical substances that help to inhibit industries to reap the benefits of this research by
the oxidation reactions caused by free radicals such as formulations containing elements reported to be essential
singlet oxygen peroxy radicals, hydroxyl radicals and for human health. A variety of such formulations are
peroxy nitrite thereby preventing or delaying damage to available worldwide (Iqbal et al., 2011).
the cells & tissues. The mechanism of action include
Ranunculus muricatus, which is commonly known
*Corresponding author: e-mail: muhammadzahoorus@[Link] as spiny fruit buttercup is a member of Ranunculaceae
Pak. J. Pharm. Sci., Vol.29, No.2, March 2016, pp.503-510 503
Chemical and biological evaluation of Ranunculus muricatus

family. About 22 genera and 114 species including the extract was concentrated by heating on a water bath
Ranunculus muricatus are available in Pakistan that are till 1/4th of its original volume. Concentrated NH4OH was
of great concern in term of medicine and is used for added till the precipitation was completed. The mixture
varieties of ailments like intermittent fevers, gout and was allowed to settle. The precipitates were collected,
asthama (Chopra et al. 1986). Although Ranunculus washed with dilute ammonium hydroxide and filtered.
muricatus is traditionally used by local practitioners for The residue obtained was alkaloids, which were then
the treatment of various ailments but still it is not dried and weighed (Hanna, 2008; Lee et al., 2003).
scientifically proved. Local practitioners and Hakeems are
not fully aware of toxicity of heavy metal so it is needed Determination of total phenols
to standardize plant materials in term of heavy metals and To determine phenols qualitatively 2ml of ethyl alcohol
inorganic constituents. The therapeutic effect is mainly was added to test solution and few drops of ferric chloride
due to the phytochemicals present in different parts of put in it and observed for yellow coloration (Hanna, 2008;
plants. Keeping the need of these vital constituents, it is Lee et al., 2003).
required to explore its biological activity and antioxidant For quantitative determination of phenols the plants
activity, which will provide scientific database for further sample was boiled for 15min with 50ml of
studies. (CH3CH2)2O.5ml of the boiled mixture was taken into a
50ml flask, and 10ml of distilled water was added to it.
In this study the whole Ranunculus muricatus was Then 2ml of NH4OH solution and 5ml of concentrated
subjected to extraction of various components. The CH3 (CH2)3CH2OH were added to the mixture. The
various extracts were evaluated for phytochemicals, mixture was made up to the mark by adding distilled
inorganic profile, antioxidant and biological activities. water and left to react for 30min for color development.
The amount of phenols was determined spectro-
MATERIALS AND METHODS
photometrically at 505nm (Hanna, 2008; Lee et al., 2003).
The selected medicinal plant was collected in the month Determination of flavonoids
of March 2012 and transferred to the laboratory for For qualitative determination of flavonoids 1.5ml of a
taxonomical identification and confirmation (Voucher No: 50% aqueous methanol was added to 4ml of plant
[Link] 508) at the Pakistan Council of Scientific and extracts, warmed gently and Mg metal was added to it.
Industrial Research (PCSIR) laboratories complex, Then 5-6 drops of concentrated HCl was added to the
Peshawar. The plant samples were rinsed with tap water resulting mixture and was observed for red coloration
followed by washing with de-ionized water. Roots stem (Hanna, 2008; Lee et al., 2003).
and leaves were separated and dried in shady shelves. The
dried materials were chopped, crushed and powdered with For quantitative determination of flavonoids, 10g of the
electrical grinder. The dried powdered samples were plant sample was added to 100ml of 80% aqueous
stored in polyethylene bottles for further use. methanol and was kept at room temperature for 24 hours.
The whole solution was filtered and the filtrates were
Phytochemical determination dried by evaporation on water bath till constant weight
The stem, root and leaves aqueous extracts were prepared (Hanna, 2008; Lee et al., 2003).
by soaking 10g of powdered samples in 200ml of distilled
water for 12h. The extracts were then filtered using Determination of saponins
Whattman filter paper. The constituent phytochemicals in For qualitative determination of saponins, to a 2ml test
each sample were determined qualitatively and solution was added 2ml of distilled water and was shaken.
quantitatively using standard methods described in The appearance of foamy lather on the surface indicates
literature (Hanna, 2008; Lee et al., 2003). the presence of saponins.

Determination of alkaloids For quantitative determination of saponins, 20g grounded

For qualitative determination of alkaloids, the extracts plant samples were mixed with 100ml of 20% aqueous
were evaporated to dryness and the residues were heated ethanol. The mixture was heated on a water bath for 4h at
with 2% HCl solution on a boiling water bath. The 55°C with continuous stirring. The resulting mixture was
extracts were cooled, filtered and then treated with the filtered. The residue was then re-extracted with 100ml of
Mayer’s reagent. The appearance of yellow precipitation 20% aqueous ethanol and was evaporated to 40ml of its
showed the presence of alkaloids (Hanna, 2008; Lee et volume by heating it on water bath at 90oC. The
al., 2003). concentrates were transferred to a separating funnel and
were shacked well with 20ml of diethyl ether. The
For quantitative determination of alkaloids, 5g of each aqueous layer was subjected to further purification while
sample was put in a beaker and 200ml of 10% CH3COOH the organic layer was discarded. To the purified aqueous
in C2H5OH was added to it. The mixture was covered and layer 30 ml of n-butanol was added and was washed twice
allowed to stand for 4h. The mixture was then filtered and with 10ml of 5% NaCl solution. The remaining solution
504 Pak. J. Pharm. Sci., Vol.29, No.2, March 2016, pp.503-510
 Farhat Ali Khan et al

was then heated on a water bath till dryness. The dried replicates were also performed for each extract against the
residues were put in oven to get a constant weight test organisms. Simultaneously, addition of the respective
(Hanna, 2008; Lee et al., 2003). solvent instead of extract was carried out as controls.
After incubation, the zones of inhibitions were measured
Determination of tannins in millimeters and mean values were calculated
For qualitative determination of tannins 0.5 ml extract (Sofowara, 1993; Chouhan et al., 2002).
solution was added with 1ml of distilled water and 1-2
drops of ferric chloride solution, observed for blue-black Preparation of standard fungal suspension
coloration. The fungal cultures, Aspergillus niger (ATCC 9763) and
Candida albicans (ATCC7596) were incubated on
For quantitative determination of tannins 500mg of saboraud dextrose agar for four days at 25ºC. The cultures
powdered plant sample was taken into a 50 ml flask. 50ml were harvested and washed with sterile saline solution
of distilled water was added to it and stirred for 1h. The and the suspension was stored in refrigerator for further
mixture was filtered into a 50ml volumetric flask and the use (Sofowara, 1993; Chouhan et al., 2002).
volume was made up to the mark with distilled water.
Pipette out 5ml of the filtered sample into test tube and Table 1: Qualitative phytochemical analysis of
was mixed with 2ml of 0.1M ferric chloride solution. The Ranunculus muricatus
amount of tannins was determined using
spectrophotometer at 395nm within 10min (Hanna, 2008; Constituent Root Stem Leaves
Lee et al., 2003). Alkaloids +ve +ve +ve
Flavonoids +ve +ve +ve
Antimicrobial activity Tannins +ve +ve +ve
Preparation of Crude Extract Saponins +ve +ve +ve
The crude extract was prepared by contacting 100g each Phenols +ve +ve +ve
of root, stem and leaves powder with 90% methanol and
kept for two weeks with continuous stirring at regular Table 2: Quantitative phytochemical analysis of
intervals. After two weeks the mixtures were filtered from Ranunculus muricatus (%)
cloth and the liquid portions were dried in rotary
evaporator. From crude extract aqueous and acetone Constituent Root Stem Leaves
fractions were prepared by dissolving it in distilled water Alkaloids 0.967 0.801 0.456
and acetone respectively and dried using rotary Flavonoids 0.403 0.699 0.313
evaporator. Tannins 0.013 0.015 0.009
Saponins 4.730 7.110 7.350
Preparation of standard bacterial suspension Phenols 0.0038 0.0044 0.0031
The average number of viable organism per ml of the
stock suspensions containing Bacillus subtilis Anti-fungal activity
(NCTC8236), Escherichia coli (ATCC25922), Well agar diffusion method was used to determine the
micrococcus luteus (ATCC6380), Pseudomonas antifungal activities of the prepared water, methanolic and
aeruginosa (ATCC27853), Salmonella typhi (ATCC0650) acetone extracts. The 0.6ml standard fungal stock
and Staphylococcus aureus (NCTC25953) were suspension 108-109 CFU/ml were mixed with 60ml of
determined by means of the surface viable counting sterile yeast and mould extract agar thoroughly. 20ml of
technique. About 108-109 colony-forming units (CFU) per inoculated yeast and mould extract agar mixture was
ml were used. Each time a fresh stock suspension was poured into sterile petri dishes. Allowed the agar to settled
prepared (Sofowara, 1993; Chouhan et al., 2002). and four wells (10mm in diameter) were made in each of
these plates using sterile cork borer No 8. Then agar discs
Antibacterial activity were removed. The entire wells were filled with 0.1ml of
The antimicrobial activities of the extracts were root, stem and leave extracts using micro titer-pipette and
determined by well agar diffusion method (Sofowara, allowed to diffuse at room temperature for 2h. The plates
1993). The standard bacterial stock suspension 108-109 were incubated at 25oC for four days. Simultaneously,
CFU/ml was mixed with 60ml of sterile nutrient agar addition of water, methanol and acetone instead of extract
thoroughly and 20ml from inoculated nutrient agar was was carried out as controls (Sofowara, 1993; Chouhan et
poured into sterile petri dishes. These were left for some al., 2002). After incubation, the zones of inhibition in
time to set. Then four wells (10mm in diameter) were millimeter were measured and mean values are presented
made in each of these plates using sterile cork borer No. 8 in table 6.
and agar discs were removed. The entire wells were filled
with 0.1ml of root, stem and leave extracts using micro Determination of physical parameters
titer-pipette and allowed to diffuse at room temperature The pH of extracts was determined by pH meter (InoLab
for 2h. The plates were incubated at 37oC for 24h. Two pH 720). The pH meter was first calibrated with two point
Pak. J. Pharm. Sci., Vol.29, No.2, March 2016, pp.503-510 505
Chemical and biological evaluation of Ranunculus muricatus

Table 3: Zones of inhibitions of water extracts of Ranunculus muricatus in millimeters of bacterial strains

Plant’s part B. Subtillis S. Aureus M. Luteus E. Coli P. Aeruginosia S. Typhi

Root 5 6 7 2 3 5
Stem 3 3 5 2 3 2
Leaves 6 8 8 4 3 3
Table 4: Zones of inhibitions of methanolic extracts of Ranunculus muricatus in millimeters of bacterial strains
Plant’s part B. Subtillis S. Aureus M. Luteus E. Coli P. Aeruginosia S. Typhi
Root 12 10 13 7 5 10
Stem 7 11 10 5 7 7
Leaves 13 12 13 8 9 8
Table 5: Zones of inhibitions of acetone extracts of Ranunculus muricatus in millimeters of bacterial strains

Plant’s part B. Subtillis S. Aureus M. Luteus E. Coli P. Aeruginosia S. Typhi

Root 8 9 10 5 5 7
Stem 5 9 6 5 4 7
Leaves 10 12 11 6 4 7
Table 6: Zones of inhibitions of water, methanolic and acetone extracts of Ranunculus muricatus in millimeters of
fungal strains

Water extract Methanolic extract Acetone extract

Plant’s part
A. Niger C. Albican A. Niger C. Albican A. Niger [Link]
Root 0 1 2 2 1 1
Stem 0 1 1 2 0 1
Leaves 1 1 1 2 1 1
buffers (pH= 4 and 9). The sample in form of juice was mixture was cooled at room temperature and was added
taken in 50ml beaker and pH electrode was inserted in it. with deionized water to keep it overnight. The
The pH was noted from screen when stabilized. After undissolved particles were filtered and the volume of the
each reading the electrode was wiped out with small piece filtrate was made up to 100 ml. The filtrates were used as
of cotton soaked in distilled water. sample solution for the determination of inorganic
constituents (Pendias, 1986).
Hardness was measured through complex metric titration
with EDTA while electrical conductivity was measured Sodium and potassium were determined by flame
using conductivity meter (HI 99300 HANNA). photometer (Coning-40). Calcium and magnesium were
determined by complexometric titration while phosphate
Determination of heavy metals was determined by calorimetric method using ammonium
For the determinations of heavy metals 1g of root, stem dihydrogen phosphatase standard solution and
and leaves samples were charred in a crucible for 4 to 6 molybodate as complexing agent. The sulphate and
minutes. After charring, ashing was done by putting plant bicarbonate were determined by titrimetric method and
samples in a furnace for 5h at 600°C in a crucible. After chloride contents were determined by the standard
ashing, it was cooled in the desiccators. The cooled argentometric method using potassium chromate indicator
contents were dissolved in 2.5ml of 6M HNO3. The (Farhat et al., 2011).
samples were filtered and the filtrates were kept in plastic
bottles for further use. The heavy metals in the samples DPPH radical-scavenging activity
were determined by atomic absorption spectrophotometer Hydrogen donating or radical scavenging ability of
(Hitachi, Model Z-8000 Japan) analysis. acetonic, chloroform and ethanolic fractions of the
extracts of Ranunculus muricatus stem, leaves and root
Determination of inorganic constituents were measured by using the table DPPH method (Hanato
The collected plant was dried at 120°C to get a constant et al., 1988). The different extracts were diluted and 1ml
weight. It was grinded to fine powder and was subjected of each diluted extract was added to 0.25ml of a 0.2
to drying ashing. For drying ashing pre-cleaned silica mmol/l DPPH methanolic solution. The mixtures were
crucible was heated at 600°C to a constant weight. The placed in dark at room temperature for 30min. Using UV-
powdered sample material was heated in a muffle furnace Visible spectrophotometer the absorbance of the resulting
at 600°C till there was no evolution of smoke. The solution was examined at 517nm. Using the following
506 Pak. J. Pharm. Sci., Vol.29, No.2, March 2016, pp.503-510
 Farhat Ali Khan et al

equation, the ability to scavenge the DPPH radical was Heavy metal contents
calculated: The heavy metals contents (mg/Kg) in Ranunculus
muricatus are given in table 8.
DPPH scavenging effect (%) =
Where A0 is the absorbance of the control at 30 min, and Inorganic constituents
A1 is the absorbance of the sample at 30 min. All samples Table 9 shows the quantitative determination of inorganic
were analyzed in triplicate. constituents of Ranunculus muricatus.

RESULTS Table 10: DPPH anti scavenging activities (%) for

chloroform, ethanolic and acetone extracts of Ranunculus
Phytochemicals analysis muricatus.
The qualitative phytochemical analysis showed the Plant’s Chloroform Ethanolic Acetone
presence of alkaloids, flavonoids, tannins, saponins and Part extract extract extract
phenols. The results of qualitative phytochemical analysis
Root 49.57 51.5 61.3
are given in table 1. The qualitative phytochemical
Stem 43.9 49.9 54.3
analysis results were further confirmed by quantitative
Leaves 52.6 53.5 66.9
phytochemical analysis (table 2).
Antimicrobial activities
The zones of inhibitions of water, methanolic and acetone Antioxidant activities
extracts in millimeter for 6 different bacterial strains are To evaluate the antioxidant activities of fruits and
summarized in tables 3-5 and for 2 fungal strains are vegetables numerous analyses such as total antioxidant
given in table 6. activity, DPPH and ABTS assays, ROS quenching assay,
metal chelating, reductive potential, β-carotene linoleate
Table 7: Physical parameters of Ranunculus muricatus system and linoleic acid method are the most commonly
used.
Names of Parameter Root Stem Leaf
pH 10.35 10.4 9.9 The antioxidant activities of Ranunculus muricatus are
EC 1142 847 576 given in table 10.
TDS 867.8 818.3 523.4 DISCUSSION
Hardness 210 70 120
Phytochemicals analysis
Table 8: Concentration (mg/Kg) of selected heavy metals Alkaloids are cyclic organic compound having nitrogen in
in Ranunculus muricatus a negative oxidation state. They have limited distribution
in living organism (Hodnick et al., 1988). Alkaloids are
Heavy metal Root Stem Leaves
used as dyes, spices, drugs or poisons. They are well
Cu 0.38 0.08 0.24
known for their CNS activities (Cook and Samman, 1996;
Fe 20.05 8.19 42.40 Yamamoto and Gaynor, 1980). High concentration of
Pb 0.0 0.0 0.07 alkaloids was found in root followed by stem and
Zn 1.12 0.50 0.80 comparatively less amount in leaves.
Table 9: Concentration (mg/Kg) of selected cations and Flavonoids are a group of polyphenolic compounds
anions in Ranunculus muricatus possessing low molecular weight that exhibit a common
benzo-pyrone structure. They show anti-allergic (Cushnie
Name of Parameter Root Stem Leaves and Lamb, 2005), anti-inflammatory (Xu et al., 2005),
HCO3-1 130 130 230 antimicrobial and anticancer activities (Oakenfull, 1986).
Ca+2 12 16 20 Flavonoids also referred as bioflavonoids, are polyphenol
Mg+2 43.74 7.29 17.01 antioxidants found naturally in plants. They are secondary
Cl-1 441.60 338.4 115.20 metabolites that have no direct involvement with the
NO3-1 6.1 12.1 4.1 growth or development of plants. The effect of flavonoids
SO4-2 13 12 33 on plants growth is indirect and associated with the action
Na+1 270 280 170 of auxins. Flavonoids can improve the blood circulation
K+1 24 33 12 and reduces the blood pressure (Zhang et al., 2001). High
F-1 0.28 0.17 0.11 amount of flavonoids, as is evident from table 2 was there
in stem followed by root and least in leaves.
Physical parameters
The various physical parameters are given in table 7. The saponins are naturally occurring surface- active
glycosides. Many pharmacological activities have been
Pak. J. Pharm. Sci., Vol.29, No.2, March 2016, pp.503-510 507
Chemical and biological evaluation of Ranunculus muricatus

reported about saponins such as antibiotic, antifungal, From table 6 it is evident that methanolic extracts showed
antiviral, hepatoprotective, anti-inflammatory and anti- highest zones of inhibition for the two fungal strains
ulcer (Hostettmann and Marston, 1995; Ireland and followed by acetone extracts. Water extracts are
Dziedzic, 1986). Saponins have been reported to have comparatively less potent against the fungal strains.
important biological activities in humans including
hypocholesterolemia, hemolytic, immunostimulatory and Physical parameters
antitumourigenic activities (Fukuda et al., 1995) as well The pH of root, stem and leaves are at alkaline side with
as chemo protective activities (Woldemichael and Wink, highest 10.35 for root extract. The electrical conductivity
2001). Steroid and triterpenoid saponins with a single was highest for root extract followed by stem extract.
sugar chain were found to have strong hemolytic activity, Highest TDS value was recorded for root extract followed
whereas those with two sugar chains showed less activity by stem extract. Hardness was high for root followed by
(Sindambiwe et al., 1998). Some saponins and sapogenin leaf extract.
s have been shown to be capable of deactivating viruses
for example purified saponin mixture from maesa Heavy metal contents
lanceolata (Buzzini et al., 2008). The comparatively high Some of the heavy metals are needed for the normal
percentage of saponins was observed in root, stem and growth of plants and animals while others are toxic to
leaves as compared to other phytochemical constituents. both of them. From table 8 it is evident that iron is present
in highest concentration (42.40mgKg-1) in leaves as
Tannins are basically use for the treatment of compared to root and stem. Iron is an essential trace
inflammation, leucorrhoea, gonorrhea, burn, piles, element required by all forms of life. In man it is required
diarrhea and as antidote in the treatment of alkaloidal for the synthesis of haem proteins which function in the
poisoning (Williamson and Manach, 2005). They are also process of oxygen transport and oxidative metabolism.
used for tanning of animal hides to convert them to The total body iron for an adult male has been estimated
leather. High concentration was there in stem followed by to be about 4g and for the female 2.5g. The requirements
root and leaves. for growth have been estimated to be 30mg/kg body
Phenols are very wide spread in nature. They range from weight. In rats an oral administration of 60-100mg/kg
simple structures having a simple aromatic ring to highly body causes toxicological effects. The iron contents
complex polymeric structures and often exist in present in the samples are below the toxic level (Odell
glycosidic forms (Mattila and Hellström, 2007). Capsaicin and Sunde, 1997).
is found in the dried ripe fruit of different species of Zinc is an essential element in the nutrition of man,
Capsicum. It has been used internally for dyspepsia and animals and plants. It acts as an integral part of numerous
flatulence. Externally it is frequently used as enzymes. Because of its essentiality, zinc is present in all
counterirritant (Cragg et al., 1999). From table 2 it is clear plant and animal tissues. The total body zinc for a 70kg
that the phenols concentration is low in the whole plant. individual has been estimated to be 2.3g. Zn contents
Comparatively high concentration of phenols was there in were very low as compared to iron contents. Highest
stem and root. concentration of Zn 1.12mgKg-1 was recorded in root
Antimicrobial activities followed by leaves and stem. The Food and Nutrition
Sustainable amount of new antibiotic available in the Board of the United States recommendations for dietary
market are obtained from natural or semi synthetic allowances for zinc are as follows: infants 0-0.5 years, 2
resources which are obtained from about 20% of the mg and 0.5-1.0 years, 5 mg; children 1-10years, 10mg;
plants present in world which were submitted to men and women 11-51+ years, 15mg; pregnant women,
pharmaceutical and biological tests. The chemical 20mg and lactating women, 25mg. Similar figs have been
compounds with antimicrobial activities isolated from recommended by WHO/FAO (Odell and Sunde, 1997;
plants have vast remedial power and are useful in the cure WHO, 2004).
of infectious diseases. Several plants byproducts possess Lead is toxic to both plants and animals. Its maximum
antimicrobial activities against pathogenic bacteria and acceptable limit in foodstuffs is 1 mgKg-1. In root and
fungi (Bylka et al., 2004). stem samples the concentration of lead was beyond the
As can be seen from table 3, high activity 8 mm was detectable limit. However in leaves its concentration was
recorded for leaves water extracts against S. Aureus and 0.07mgKg-1which is below the acceptable limit for food
M. Luteus while less activity 3mm was seen against P. stuffs (Odell and Sunde, 1997).
Aeruginosia and S. Typhi. The leaves extracts showed
highest inhibition zones as compared to root and stem Copper is an essential component of certain enzymes and
extracts. Same trend was observed for methanolic and is required for normal growth and development of plants
acetone extracts tables 4 and 5). Methanolic extracts and animals. The essential role of copper in maintaining
exhibit highest zones of inhibitions followed by acetone normal health in both animals and humans has been
extracts as compared to water extract. recognized for many years. The average daily dietary
508 Pak. J. Pharm. Sci., Vol.29, No.2, March 2016, pp.503-510
 Farhat Ali Khan et al

requirement for copper in the adult human has been lead concentration in the samples was below the
estimated at 2 mg and for infants and children at acceptable limit for the food stuffs (1mgKg-1 body
0.05mg/kg body weight (WHO, 2004). weight). The physical parameters, antioxidant and
antimicrobial activities of the extracts were also
It can be toxic at excessive level. Phytotoxicity can occur determined. Highest antioxidant activities was observed
at a concentration higher than 20mgKg-1 of the dry weight for acetone extract fraction as compared to ethanolic and
of the plant. The concentration of copper in Ranunculus chloroform extract fractions. Highest zone of inhibitions
muricatus is below the phytotoxic level. were observed for methanolic extract followed by acetone
extract fraction against micrococcus luteus. Antifungal
Inorganic constituents activities were high for methanolic extracts against
All inorganic constituents shown in table-9 are vital to Candida Albican. Phytochemicals, antioxidant,
growth of the plant and animals. Some of them are growth antimicrobial and minerals analysis in the present study
initiators while others are enzyme activators. Calcium showed that Ranunculus muricatus can be used as
strengthens the bones and its importance almost becomes medicine due to its high antioxidant, antimicrobial
double during pregnancy. It also helps in blood activities, presence of essential phytochemical and
coagulation. Potassium activates certain enzymes, inorganic constituent.
whereas iron is integral part of hemoglobin. Inorganic
constituents are essential in one way or the other to life ACKNOWLEDGEMENT
and their importance cannot be neglected at any level.
We are thankful to Higher Education Commission (HEC),
Bicarbonates and Ca+ were present in leaves while high Pakistan for financial support No. PM-IPFP/HRD/HEC/
concentration of Cl-1 and Mg+2 were observed in root of 2011/3393
the plant. NO3-1 and SO4-2 amount were high in stem and
leaves respectively. Na+1 and K+1 content were high in REFERENCES
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