Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502

Vol. 2(ISC-2012), 12-15 (2013) Res. J. Recent. Sci.

Phytochemical analysis of leaf extract of Phyllanthus fraternus

Mehta Kavit1, Patel B.N.1 and Jain B.K.2
1
Mehsana Urban Institute of Sciences, Ganpat University, Ganpat Vidyanagar, Kherwa, Dist. Mehsana, North Gujarat, INDIA
2
M.G. Science Institute, Navrangpura, Ahmadabad, INDIA
Available online at: www.isca.in
Received 17th October 2012, revised 29th January 2013, accepted 07th February 2013

Abstract
Phyllanthus fraternus is a pan tropical weed and probably originates from western India. This plant belongs to
Euphorbiaceae family. In India it is used as a herbal medicine and called as ‘Bhumyamlaki’. It is a large genus comprising
about 750 species in tropical and subtropical regions. The leaves of Phyllanthus fraternus are collected from botanical
garden of University campus. The leaves are extracted in chloroform solvent and evaluated for phytoconstitutes present in
them. For phytochemical analysis of plant extract thin layer chromatography and preliminary screening method of
phytoconstitute by Sofowara, Trease and evans and Harbone was followed. The plant extract contains alkaloids like
morphine and boldine. Extract also contains tannins, saponin, terpenoid and steroid. The present study provides evidence
that solvent extract of Phyllanthus fraternus contains medicinally important bioactive compounds and this justifies the use of
plant species as traditional medicine for treatment of various diseases.

Keywords: Morphine, borbine, berbeline, tannin.

Introduction niranthin, nirtetralin and phyltetralin and other compounds like

alkamide, alkaloid, terpenoid, flavnoid2.
India has one of the oldest, richest and most diverse cultural
traditions associated with the use of medicinal plants. This However, there are no reports on phytochemical analysis of leaf
knowledge is accessible from thousands of medical texts and extract of P. fraternus. Hence, the authors have made an attempt
manuscripts. This traditional knowledge forms the codified on phytochemical analysis of leaf extract which was followed
system s of medicine and exists in the forms of Ayurveda, by thin layer chromatography.
Unani, Siddha and Swa-riga (Tibetan) systems of medicine. The
flora and fauna are used for medicinal purposes and they have Material and Method
important cultural roles and as well as vital roles in forest
ecology, such as pollination, seed predation and dispersal, seed Collection of plant: Fresh plant leaves of Phyllanthus fraternus
germination, herbivory and predation on potential pest species. were collected from Botanical garden, S. K. Pharmaceutical
college of education and research; Ganpat University; Ganpat
Ethnomedicinal study deals with the study of traditional vidyanagar, Kherwa (Mehsana, Gujarat).The leaves were
medicines. Since ancient times mankind has been using herbal washed thoroughly with normal tap water followed by sterile
plants, organic materials as well as materials from the sea, rivers distill water. Then leaves were dried under shaded condition at
etc. for its betterment. These substances have been used as food, room temperature. Leaves were crushed to powder using
medicine etc. Amongst them, the substances having medicinal grinding machine. Powder were stored at 4 oC in tight air
value have been extensively used for treating various disease container bottle.
conditions. Herbs being easily available to human beings, have
been explored to the maximum for their medicinal properties. Sample preparation for phytochemical screening: 50 gm
Various parts of the plants like roots, leaves, bark, exudates etc. powdered sample was weighed and taken separately. The
are used as per medicinal properties1. powder was moisten with ammonia and evaporated to dryness.
Dried sample was extracted with chloroform and filtered. After
Phyllanthus fraternus is a pantropical weed which is probably filteration, extract the chloroform layer with 10% sulfuric acid
originated from western india. This plant belongs to using separating funnel. and separate aqueous layer adjust with
Euphorbiaceae family.In India it is used as a herbal medicine pH 8 with ammonia ; after adjusting pH extract this solution
and called ‘Bhumyamlaki’. It is a large genus comprising about with chloroform which organic extract obtained were evaporate
750 species in tropical and subtropical region. Phyllanthus to concentrate by kept open room temperature. However
amarus, a member of the same family was studied for its Aqueous extraction was evaporated to dryness by heating in
phytochemical analysis and it was shown that it contains lignans waterbath to obtain semi solid mass. Dried extract was stored in
refrigerator for their future use in phytochemical analysis.

International Science Congress Association 12

Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502
Vol. 2(ISC-2012), 12-15 (2013) Res. J. Recent. Sci.

Phytochemical screening: Chemical tests were carried out sample using Bandwise with Linomat 5 (camag, muttez;
using aqueous extract to identify various constitutes using Switzerland) spray on automated instrument for HPTLC.
standard methods of Sofowara, Trease and evans and Harbone. Applied sample band length 8 mm 4 track, track distance 15
mm, distance from lower edge 15mm;application volume 1-20μl
Test for Alkaloid: 3 ml aqueous extract was stirred with 3 ml of sample at 4 track. camag twin through chamber with
of 1% HCl on steam bath. Mayer and Wagner’s reagent was Toluene-chloroform-ethanol 4:4:1 after20 min pre-saturation
than added to mixture. Turbidity of the resulting precipitate was with mobile phase for development were used. The four
taken as an evidence for the presence of alkaloid development over 62.9 mm with intermediate drying after the
run plate were dried and heated at 110oC for 1 hr for detection
Test for Tannins: About 2 ml of the aqueous extract was of active compound.
stirred with 2 ml of distilled water and few drops of FeCl 3
Solution were added. Formation of green precipitate was The camag TLC Scanner 3 controlled by win CATS software
indication of presence of tannins. was used for densitometry analysis. For this densitometry
analysis observed Absorption measurement at 254,366 and 540
Test for Saponins: 5 ml of aqueous extract was shaken nm with TLC Scanner 3 controlled by win CATS software.
vigorously with 5 ml of distilled water in a test tube and
warmed. The formation of stable foam was taken as an Determination of constitutes by TLC: For TLC analysis Plate
indication of the presence of saponins. with aluminum support silica gel60F254, 10X10 cm(merck) were
cut with ordinary household scissors. plate markings were made
Test for Phlobatannins: About 2 ml of aqueous extract was with soft pensil. Silica gel plate preparation plate impregnated
added to 2 ml of 1% HCl and the mixture was boiled. by dipping into 4 % solution of sodium acetate in methanol –
Deposition of a red precipitate was taken as an evidence for the water 3:2 for 5s followed by drying at room temperature for 1 hr
presence of phlobatannins. .Glass capillaries were used to spot the sample for TLC applied
sample volume 1-μl of sample by using capillary at distance of 1
Test for Flavonoids: To 1 ml of aqueous extract, 1 ml of 10% cm at 3 track. In the twin trough chamber with Toluene-ethyl
lead acetate solution was added. The formation of a yellow acetate-diethyl amine 7:2:1 after pre-saturation with mobile
precipitate was taken as a positive test for flavonoids. phase for 20 min for development were used. Three
developments over intermediate drying. After the run plates are
Test for Terpenoids: 2 ml of the organic extract was dissolved dried and sprayed dragndorff reagent at room temp for 10- 15
in 2 ml of chloroform and evaporated to dryness. 2 ml of min for detection of active compound.
concentrated sulphuric acid was then added and heated for about
2 min. Development of a greyish colour indicates the presence Result and Discussion
of terpenoids.
The results confirm the presence of constituents which are known
Tests for glycosides: Liebermann’s test: 2 ml of the organic to exhibit medicinal as well as physiological activities3. The
extract was dissolved in 2 ml of chloroform and then 2 ml of phytochemical characteristics of the leaf extract of Phyllanthus
acetic acid was added in it. The solution was cooled well in ice. fraternus investigated are summarized in table-1.
Sulphuric acid was then added carefully. A colour change from
violet to blue to green indicates the presence of a steroidal The results reveal the presence of medicinally active
nucleus (that is, a glycone portion of glycoside). constituents like tannins, Alkaloid, terpenoids, steroids and
saponins in the leaves of Phyllanthus fraternus. While Flavnois,
Tests for steroids: i. A red colour produced in the lower Phlobatannins, Glycosides were absent in this plants.
chloroform layer when 2 ml of organic extract was dissolved in
2 ml of chloroform and 2 ml concentrated sulphuric acid was Determination of constitutes by HPTLC summarized in figure1
added in it, indicates the presence of steroids. ii. Development b,c and d showed that under 256 nm and 366 nm only
of a greenish colour when 2 ml of the organic extract was chlorophyll was observed while after 1 hr at 110oC treatment
dissolved in 2 ml of chloroform and treated with sulphuric and under 540 nm a orange brown band was observed which
acetic acid indicates the presence of steroids. indicated presence of alkaloid.

Determination of constitute by HPTLC: For HPTLC different Determination of constitute by TLC summarized in figure 1 a.
HPTLC plate were used. Plates with aluminum support silica showed that after drying spray with dragndorff reagent for 10 -
gel60F254, 10X100 cm (merck) were cut with ordinary 15 min a brown band was observed which indicated presence of
household scissors. plate markings were made with soft pensil. alkaloid.
Silica gel plate preparation plate impregnated by dipping into 4
% solution of sodium acetate in methanol – water 3:2 for 5s The alkaloids contained in plants are used in medicine as
followed by drying at room temperature for 1 hr and spot the anaesthetic agents4. The presence of saponins in plants have

International Science Congress Association 13

Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502
Vol. 2(ISC-2012), 12-15 (2013) Res. J. Recent. Sci.

been reported to be responsible for the tonic and stimulating

activities observed in Chinese and Japanese medical herbs5. The
results obtained in this study thus suggest that the identified
phytochemical compounds may be the bioactive constituents
responsible for the efficacy of the leaves of the plants studied.
The presence of some of these compounds have also been
confirmed to have antimicrobial activity6. Hence it could be
inferred that the plant extracts could be a source for the
industrial manufacture of drugs useful in the chemotherapy of
some microbial infection.

Table 1
Phytochemical constitute of the leaf extract of Phyllanthus
fraternus
Chemical Constitutent leaf extract of Phyllanthus (c) HPTLC at 254 nm
fraternus
Alkaloid Present
Tannins Present
Saponins Present
Phlobatannins Absent
Flavanoid Absent
Terpenoid Present
Glycosides Absent
Steroid Present

(d) HPTLC at 366 nm

Figure-1
Determination of constitute by TLC and HPTLC
(a)TLC (b) HPTLC at 540 nm (c) HPTLC at 254 nm (d)
HPTLC at 366 nm

Conclusion
Phyllanthus fraternus plant belongs to Euphorbiaceae family. It
(a)TLC
is used as a herbal medicine and called as ‘Bhumyamlaki’. there
are no reports on phytochemical analysis of leaf extract of P.
fraternus. Author investigated and collected leaves of
Phyllanthus fraternus from botanical garden of University
campus. The leaves are extracted in chloroform solvent and
evaluated for phytoconstitutes present in them. For
phytochemical analysis of plant extract thin layer
chromatography and preliminary screening method of
phytoconstitute by Sofowara,Trease and evans and Harbone was
followed. The plant extract contains alkaloids like morphine and
boldine. Extract also contains tannins, saponin, terpenoid and
steroid. The present study provides evidence that solvent extract
of Phyllanthus fraternus contains medicinally important
bioactive compounds and this justifies the use of plant species
(b) HPTLC at 540 nm as traditional medicine for treatment of various diseases.

International Science Congress Association 14

Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502
Vol. 2(ISC-2012), 12-15 (2013) Res. J. Recent. Sci.

Reference 6. Alagesaboopathi C. and Sivakumar R., Antimicrobial

properties of various extracts of andrographis neesiana
1. Perumal Samy R. and Gopala Krishnakone P., Current wight-an endemic medicinal species from india,
status of herbal and their future perspectives, Nature International Journal of pharmatech research, 3(1), 27-31
Precedings: hdl:10101/npre.1176.1: (2007) (2011)
2. Akinjogunia O.J., Eghafona N.O., Enabulele I.O., Mboto 7. Trease G.E. and Evans W.C., Pharmacognosy, 11th edn.,
C.l. and Ogbemudia F.O., Antibacterial activity of Bailliere Tindall, London, 45-50 (1989)
ethanolic extracts of phyllanthus amarus against extended
spectrum β-lactamase producing Escherichia coli isolated 8. Usher G., A Dictionary of Plants Used by Man. C.B.S
from stool samples of HIV sero-positive patients with or Publishers and Distributors, Delhi-110032, India 220
without diarrhoea, African Journal of Pharmacy and (1984)
Pharmacology, 4(6), 402-407 (2010) 9. Morabandza C.J., Ongoka R.P., Matini L., Epa C.,
3. Ismaila Y. Sudi, Denban M. Ksgbiya1 and Emmanuel K. Nkounkou L.C. and Abena A.A., Chemical composition of
Muluh1, Nutritional and phytochemical screening of Senna the Mesocarp of Garcinia kola Heckel (Clusiaceae) Fruit,
obtusifolia indigenous to Mubi nigeria, Advances in Res. J. Recent Sci., 2(1), 53-58 (2013)
Applied Science Research, 2(3), 432-437 (2011) 10. Jain Monica, Rajput Ritika and Mishra Anamika,
4. Mukeshwar Pandey, Mousumi Debnath and Etal, Enhancement of secondary Metabolite Biosynthesis in
Phytomedicine: An ancient approach turning into future Bacopa monnieri: An in vitro Study, Res. J. Recent Sci.,
potential source of therapeutics, Journal of 2(1), 13-16 (2013)
Pharmacognosy and phytotherapy, 3(1), 113-117 (2011) 11. Rane Rajeshree, Patil Sushma,Gangolli Divya and
5. Victor Njoku O. and Chidi Obi, Phytochemical Ingawale Kanchan, The ash and Iron Content in Apple
constituents of some selected medicinal plants, African Juice Concentrate Powder, Res. J. Recent Sci., 1(10), 59-62
Journal of Pure and Applied Chemistry, 3(11), 228-233 (2012)
(2009)

International Science Congress Association 15