ailments with the help of plants having medicinal values is constantly a
noticeable characteristic of Islamic training. Different Surahs in the Holy
Quran such as in Al-Momeenoon, Al-Rehman, Al-Bakra and Al-Inaam narrating the
significance of medicinal plants. Islamic medicine begins with Hazrat Adam
(A.S.) and was ended at Hazrat Muhammad (SAW) but pharmaceutical search and
gathering of these medicines is still continued throughout the world . Natural
source such as plants have always been a noteworthy role in discovery and
production of new pharmaceuticals which are clinically useful in future. They
may always been a source of making fully synthetic drugs .
Ranunculus muricatus (Figure 1) belong
to genus Ranunculus that possesses more than 600 species. It is distributed
throughout the northern hemisphere and southern temperate regions in the tropic
where they usually limited to higher altitude. The most common use of
Ranunculus species in traditional medicines are anti-rheumatism, intermittent
fever and rubefacient. It has been found that the constituents that produce
these affects are due to Protoanemonin, anemonin . Traditionally it was used
against plague, abscess and tumors of plague . Whole plant is also used in
periodic fever and asthma . Ranunculus
pseudo-muricatus Baltter & Hallb is the synonym of Ranunculus muricatus . It is also known as spinyfruit buttercup
and its vernacular name is Jal dhania. Commonly it is also known as Latokari,
Kor gandal .
The aim of
the present paper is phytochemical assessment of the aerial parts of Ranunculus
muricatus for the determination of groups which are pharmacologically active.
Figure 1: Ranunculus muricatus: Sketch of aerial parts & flower
MATERIAL AND METHOD
Rotary Evaporator, Extraction
bottle, Dichloromethane (DCM), Methanol, Ultrasonic bath, Dragendorff’sreagent,
dilute ammonia solution, separating funnel, chloroform, acetic acid, Mayer’s
reagent, carbontetrachloride (CCl4), Five silica gel 60 F254 TLC
plates (20x20cm) (Merck),
Collection of Plant material:
Ranunculus muricatus was
collected from Jallo pind, Lahore as identified by Dr. Altaf Hussain Dasti,
Professor, Institute of pure and applied Biology, Bahauddin Zakariya
University, Multan giving the Catalog number 271-stw. Total wet weight of plant
collected was 15kg. It was then reduced to 5 kg of dried plant. The plant was
then ground till it become powder. The total weight of powder drug was 1 kg and
the technique for extraction for finely ground plant material. Measured
quantity of plant material (200grams) was taken in a glass bottle. After that
quantified volume of dichloromethane was added to it with constant sonication
in ultrasonic bath. It takes 24 hours to be settle down and then filtration was
performed. Repeat the process three times with dichloromethane and then
methanol. The Dichloromethane used during first, second and third soaking was
900 ml, 450ml and 250 ml respectively and 700 ml, 300ml and 250 ml for methanol
respectively. Rotary evaporator was used
for the concentration of both extracts under reduced pressure labeled with
codes as RMAPD and RMAPM respectively.
Test for Alkaloids:
(0.5-1g) was boiled with dilute hydrochloric acid (10 ml) in a test tube for
one minute; it was allowed to cool and fragments to settle down. Filtered the
supernatant liquid into other test tube. Pour three drops of Dragendorff’s
reagent. Clear precipitate or turbidity seemed, representing the occurrence of
alkaloids. To confirm the existence of alkaloids, the remaining part of
solution was made alkaline to litmus paper with dilute NH3 solution.
It was then extracted with CHCL3 (5ml) by shaking it gradually and
permit the layers to isolated. The lower CHCL3 layer was detached
and extracted with dilute CH3COOH (10 ml). The CHCL3
layer was cast-off. The extract was distributed into 4 parts and adds few drops
of Wagner’s reagent, Mayer’s reagent and Dragendorff’s reagent separately to
each of 3 parts while the 4th parts worked as untreated control. An observation
of turbidity or precipitate compared with untreated control with either or all
reagents confirmed occurrence of alkaloids .
Test for Anthraquinone glycosides:
(0.1g) was extracted with hot H2O (10 ml) for five minutes. Filter
the solution when it was hot. Cool afterward and extracted with CCl4
(10ml). The CCl4was detached, washed with water (5ml) and shaken
with dilute NH3 solution (5ml). Absence of pink to cherry red color showed the
absence of free anthraquinone Ground drug (0.1g) was dissolved with Iron (III)
chloride (10 ml) and HCl (5ml). ? the solution on heated bath for ten minutes.
Filter the solution when it was hot. Cool afterward and extracted with carbon
tetrachloride (10ml). The carbon tetrachloride was detached, washed with water
(5ml) and shaken with dilute NH3 solution (5ml). Absence of pink to
cherry red color showed the absence of bound anthraquinone .
Test for Cardioactive glycosides:
Keller Kiliani test:
Drug used for
analysis was crushed then taking 1g. 10 ml of alcohol (70%) with 1g of crushed
drug was boiled on water bath for two minutes. Filter the extract and add
distilled water (twice amount) to dilute the filtrate. After then lead sub
acetate was added. Filter it. CHCL3 or CCL4 was used for
extraction of filtrate by vigorous shaking. Transfer the organic portion in a
crucible. Evaporate the organic portion and add 3ml of Iron (III) chloride (3.5
%) in a residue. Transfer the portion in a test tube and then H2SO4
was added cautiously along the wall of test tube .
Test for Tannins:
Lead acetate test:
Powder of plant was dissolved in distilled water and boiled. After
boiling filter the solution and added lead acetate in the filtrate which gave
precipitate, are indicating the presence of tannin.
Test for Saponin:
Grounded drug (0.5g) was shaken
with H2O. Consistent foam showed presence of saponin .
Thin Layer Chromatography
Test samples, organic solvents
(chloroform, methanol, ethyl acetate, n-hexane and isopropyl alcohol), Spotting
capillary, coated TLC plates, TLC tank, oven and UV illuminator.
Spotting and Development of TLC plates:
Ten mg of
each methanolic and dichloromethane extracts of aerial parts were dissolved in
1ml of methanol and dichloromethane (HPLC grade), respectively. Five silica gel
60 F254 TLC plates (20x20cm) were marked at 1cm from each side and cut into
smaller plates. 5-10ul of sample was applied by capillary on the line marked.
Samples were applied at equal distance for simple TLC and spot was no more than
6mm in diameter.
Visualization of TLC plates:
were first visualized in the UV light (254nm and 366nm) and visualized spots
were marked for the determination of Rf value. Subsequently sprayed with the
was prepared by mixing equivalent volumes of 1% vanillin in ethanol and 3%
perchloric acid in water. TLC plates were sprayed with this mixture and then
with 10% sulphuric acid in ethanol. Sprayed TLC plates were then heated at
100C°. Different spots were observed .
Measurement of Rf value:
distances, covered by mobile phase and substances were measured and Rf value
Rf = Distance
traveled by the component / distance traveled by the solvent front
extraction of Aerial part of Ranunculus
muricatus maceration process was adopted. The solvent used for extraction
were methanol and dichloromethane. The results are shown in the table 1.
Phytochemical analysis of crude extract:
Phytochemical studies were carried out for detection
of secondary metabolites i.e. alkaloids, anthraquinone glycosides, cardiac
glycosides, saponins and tannin; in plant material. The results of the study
are shown in table 2.
Results of extraction of plant material with different solvents.
Extract obtained (gm)
Results of identification of secondary metabolites in crude extract.
+++ = Strongly
positively results - = No
Result of TLC of dichloromethane extract of Ranunculus muricatus:
UV active components were
observed at 254nm with Rf values as follows:
0.28, 0.39, 0.54, 0.67, 0.77, 0.82, 0.87.
components were observed at 366nm with Rf values as follows:
0.28, 0.39, 0.54, 0.67, 0.87
components became visible is Purple “P.U” following Godin reagent and 10%
sulfuric acid spray having Rf values given below:
photograph of developed TLC plate of dichloromethane extract of Ranunculus
Result of TLC of methanol extract of Ranunculus muricatus:
components were observed at 254nm with Rf values as follows:
components became visible i.e. P. U and P. I following Godin reagent and 10%
sulfuric acid spray respectively having Rf values given below:
0.46, 0.69, 0.80
of developed TLC plate of methanol extract of Ranunculus muricatus.
Current investigation deals with the phytochemical
evaluation of Ranunculus muricatus
(Ranunculaceae). The end result of phytochemical analysis of secondary
metabolites displayed the occurrence of cardiac glycosides, tannin and saponin
in aerial part of crude extract of Ranunculus
There have been reports of the presence
of alkaloids, anthocyanin, carbohydrates, coumarins, Jbenolie and phytosterols
in ethanolic extract of Ranunculus
muricatus . In comparison with the crude extract of Ranunculus muricatus under current investigations it has been found
that these constituents were reported for the very first time which can be
explored for further phytochemical studies and isolation of cardioactive
glycosides. Thin Layer Chromatography (TLC) has been a good analytical
technique for isolation and identification of the various compounds. Number of
UV visible components from dichloromethane and methanolic extract of aerial
parts of Ranunculus muricatus has been identified
through their Rf value. It has been found the difference in Rf value in
comparison between methanolic and dichloromethane extract.
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