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Detection of Pyrrolizidine Alkaloids in A Few Medicinal Plants

Conference Paper · December 2008

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Kotresha Sekharappa Katrahalli

Karnatak Science College, Dharwad
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ETHNOBOTANY & TAXONOMY OF
ANGIOSPERMS

2008

Edited by: Alka Chaturvedi

 © Rashtrasant Tukadoji Maharaja Nagpur University

First Published: December, 2008

Published By:

Subhash Belsare
Rigistrar,
Rashtrasant Tukadoji Maharaja Nagpur University

Chhatrapati Shivaraj Maharaj Administrative Premises,

Opp. Maharaj Bagh, Ravindranath Tagore Marg,
Rashtrasant Tukadoji Maharaja Nagpur University, NAGPUR – 440 106

Satish Niphadkar
Manager,
Rashtrasant Tukadoji Maharaja Nagpur University

Chhatrapati Shivaraj Maharaj Administrative Premises,

Opp. Maharaj Bagh, Ravindranath Tagore Marg,
Rashtrasant Tukadoji Maharaja Nagpur University, NAGPUR – 440 106
 Detection of Pyrrolizidine Alkaloids in
A Few Medicinal Plants 23
Kotresha K., *Shylaja R. & **Savita K.

Floristic and Taxonomy Division, Department of Botany,

Karnatak Science College, DHARWAD, Karnataka – 580 001
Email; [email protected]

*Smt.R. Shailaja, NMKRV College for Women, Jayanagara III Block, BANGALORE

** Smt. Savitha K. Department of Crop Physiology, University of Agricultural Science, DHARWAD

ABSTRACT

Pyrrolizidine alkaloids are carcinogenic and hepatotoxic. Detection of these alkaloids has been done
on Aegle marmelos Corr., Azadirachta indica A. Juss. Cassia tora L., Catharanthus roseus (L.) G.
Don., Eugenia jambolana Lam., Phyllanthus emblica L., Phyllanthus fraternus Webst. &
Tinospora cordifolia Hiers. Positive results were obtained in all the species except Eugenia
jambolana Lam., & Phyllanthus fraternus Webst. Plant materials were collected from various places
in and around Bangalore, and identified with the help of flora (Hooker 1878, Gamble 1935,
Ramaswamy & Razi 1973, Saldanha 1984, 1996). The pyrrolizidine alkaloids were detected by
spraying different reagents, Dagendroff, Ehrlich’s reagent A & B.

Key Words: Pyrrolizidine, Alkaloids, Carcinogenic, Hepatotoxic, Aegle, Azadirachta, Cassia,

Catharanthus, Eugenia, Phyllanthus, & Tinospora

Introduction:

Plants containing pyrrolizidine alkaloids are of wide distribution. The most important genera
are Senecio belonging to the family Astraceae (180 species), Crotalaria belonging to the family
Fabaceae (70 species) and Heliotropium which comes under the family Boraginaceae
(271 species). Among the families, Pyrrolizidine alkaloids containing genera are more in
Boraginaceae (28 genera). The most current lists of investigated species, however indicate that
only about 10% of the genera has thus for been studied. Most of these contain hepatotoxic or
corcinogenic pyrrolizidine alkaloids or both, and a number of these hepatotoxic alkaloids have
been shown to be mutagenic. Despite the display of hepatotoxic and carcinogenic activity, some
have also been shown activity of therapeutic value.

Materials and Methods:

Plant Materials: Plant material for the present investigation was collected from frequent
field trips to various places in and around Bangalore. The collected plant specimens were
identified with the help of Flora (Hooker 1878, Gamble 1935, Ramaswamy & Razi 1973,
Saldanha 1984). All the specimens studied were deposited as voucher specimens in the
Herbarium of Applied Botany, NMKRV College for Women, Bangalore. The details about the
place of collection, voucher number of the specimen and other are tabulated in table - 1.
 248 ETHNOBOTANY & TAXONOMY OF ANGIOSPERMS

Drying and Powdering of Plant Materials: The phytochemical investigations were carried
out only on selected parts of the collected plants (details given in table – 1). They allowed to
drying at room temperature (28  2C), care was taken to avoid fungal infection. The dried parts
were powdered by grinding with a mix-blender.
Preliminary Alkaloid Tests: 10g of air-dried sample was taken in a Soxhlet apparatus
and extracted with hot 98% methanol for six hours. The solvent was evaporated to dryness. The
residue was treated with 2N HCl, homogenized and filtered (washed with diethyl ether). Thus
obtained acidic extracts were subjected to preliminary alkaloid tests (Sarath et al, 1981). The
extract was divided into three parts. To the first part, Wagner’s second part Mayer’s and to the
third part Dagendroff reagents were added. The precipitate or turbidity that was formed with any
one reagent or all reagents, it confirms the presence of alkaloids.
Extraction of Pyrrolizindine Alkaloids: 50g of the powder of respective plants were
taken in the thimble and it was transferred into the extractor of Soxhlet apparatus and extracted
with 98% methanol for 18 hours or until colourless at the constant temperature (40 t0 60C).
The extracts were evaporated to dryness on water bath. The obtained dry sample was
acidified with dilute aqueous acid (2N HCl), homogenized, filtered and washed with chloroform
(or dichloromethane) to remove the natural organic compounds such as chlorophylls, lipids etc.,
using a separating funnel. The acidic solution was then filtered and made base with concentrated
ammonia. Then the alkaloid mixture was extracted with chloroform (dichloromethane or diethyl
ether). The aqueous layer was discarded. The chloroform layer contains the crude pyrrolizidine
alkaloid mixture (David J. robins, 1993). This was subjected for further chromatographic studies.
Preparation of Chromatographic (TLC) Plats:Silica gel G slurry was spread
uniformly on clean 20 x 20 cm plates with the help of spreader, plates were dried in hot oven at
110C.
Loading and Development of Chromatogram: Approximately 0.3 ml of each plant
extract loaded on the TLC plates using a lambda or micropipette.
The solvents used for the separation of pyrrolizidine alkaloids are ethyl acetate, acetone,
ethyl alcohol and concentrated ammonia in the ratio of 5:3:1:1 (Mattocks, 1986), stand for an
hour to saturate the chamber. Loaded plate was placed vertically in the solvent, once the solvent
has reached about 18cm; plates were removed from the chamber and dried.
Detection: The pyrrolizidine alkaloids are detected by spraying different reagents
namely, 1. Dragendorff reagent 2. Ehrlich’s reagent ‘A’ and 3. Ehrlich’s reagent ‘B’.
The modified Dagendroff reagent by Munier (1953) produces orange red colour,
Ehrlich’s reagent ‘A’: The TLC plates treat with Hydrogen Peroxide, Acetic anhydride, then
Ehrlich’s reagent ‘A’. For Ehrlich’s reagent ‘B’: The TLC plates treat only with acetic anhydride
than Ehrlich’s reagent ‘B’ by omitting initial oxidation step in both the cases magenta colour
formed (Mottocks & Jukes, 1987). hRf values were calculated using the following formula.
Distance moved by the solute from the origin X 100
hRf
Distance moved by the solvent from the origin
Results and Discussion:
The present phytochemical observations were carried out on eight species viz; Aegle
marmelos Corr. (Leaf), Azadirachta indica A. Juss. (Leaf), Cassia tora L. (Root, Stem, Leaf, Fruit
and Seed), Catharanthus roseus (L.) G. Don. (Leaf), Eugenia jambolana Lam. (Bark),
Phyllanthus emblica L. (Fruit), Phyllanthus fraternus Webst. (Stem) and Tinospora cordifolia
Hiers. (Stem). These plants are commonly used in Ayurveda for the preparation of Ayurvedic
 DETECTION OF PHYRROLIZIDINE………..………………………FEW MEDICINAL PLANTS 249

(diabetic) formulations. All the eight species were subjected to preliminary tests for alkaloids and
detection of pyrrolizidine alkaloids. In Cassia tora, five parts were considered to check the
pyrrolizidine alkaloids confined to a particular part or present in all the parts. Where as
Phyllanthus fraternus from different locations were considered for the same purpose.
For the preliminary tests, extracts of all eight plants were treated with Mayer’s, Wagner’s
and Dagendroff reagents. These tests were showed positive results with all the studied species
in one or the other reagents. In Aegle marmelos & Azadirachta indica showed positive results
with Wagner’s and Dagendroff reagents, negative with Mayer’s reagent. Cassia tora (root) gave
positive results with Mayer;s and Dagendroff reagents. Cassia tora (fruit) and Catharanthus
roseus showed strongly positive results with all the reagents used. In case of Eugenia jambolana
and Tinospora cordifolia (strongly positive with Wagner;s reagent) positive results were obtained
with Mayer’s and Wagner’s reagents and negative with Dragnedorff. Where as in Phyllanthus
emblica gave positive results with Dragnedorff reagent and negative results with Mayer and
Wagner’s reagents. In Phyllanthus fraternus (stem1 and stem2) and Cassia tora (stem, root and
leaf strongly positive) showed positive results with Wagner’s reagent, with the other two reagents
negative response was noticed. Detailed observations are tabulated in table - 1.
For the detection of pyrrolizidine alkaloids, the chromatograms were observed after
spraying with modified Dagendroff, Ehrlich’s A and Ehrlich’s B reagents. Eugenia jambolana
and Phyllanthus fraternus gave negative response with all the reagents. Whereas Catharanthus
roseus showed positive results with all reagents having different hRf values, with Dagendroff
reagent three bands i.e. 40,60 and 99 hRf values with Ehrlich’s reagents A and B only one band
was obtained in each with 91 and 69 hRf values respectively. The remaining species responded
either with one or two reagents. Cassia tora (except seed) and Phyllanthus emblica showed
bands with one of the reagents. Cassia tora stem and fruit responded with Ehrich’s reagent A
having 13 and 68 hRf values respectively, leaf with Dagendroff reagent of 99 hRf value.
Whereas root with Ehrlich’s reagent B having hRf value 93 and Phyllanthus emblica responded
with Dagendroff reagent having 92 hRf value. In case of Aegle marmelos, Azadirachta indica,
Cassia tora (seed) and Tinospora cordifolia positive results were obtained with two of the
reagents. Azadirachta indica and Tinospora cordifolia showed response with Dagendroff and
Ehrlich’s reagents B having one band each with hRf value 77,89,98 with Dagendroff and 83,
17 with Ehrlich’s ‘B’ respectively. Whereas in Cassia tora (seed) three bands hRf values 55, 75
and 99 with Dagendroff reagent and one band with hRf value 68 in Ehrlich’s reagent ‘B’ were
seen. Aegle marmelos gave positive results with Dagendroff and Erlich’s reagent ‘A’
having hRf value 96 and 29 respectively. Detailed data tabulated in table-1.
Conclusions:
In our investigation except Eugenia jambolana and Phyllanthus fraternus remaining all
other species have shown to contain pyrrolizidine alkaloids. But in case of preliminary tests
positive results were seen in Eugenia jambolana and Phyllanthus fraternus. So it is clearly
indicates that the pyrrolizidine alkaloids are absent and may be the other types of alkaloids are
present. In Cassia tora five parts were subjected to test for pyrrolizidine alkaloids and it was
found that all the parts containing pyrrolizidine alkaloids. Similarly Phyllanthus fraternusi from
different locations was considered and obtained negative results. The obtained results were
compared and conformed to David J. Robin 1993 on Phyllanthus species and Sarath et al., 1981
on Cassia species.
Since pyrrolizidine alkaloids are hepatotoxic and carcinogenic, it is very essential to check
their presence (when the Ayurvedic doctors recommending the pyrrolidizine containing drug for
heavy dosage or prolonged period of dosage) in the plant before use. If present, hepatotoxicity
should be observed and these compounds should be separated by modern chromatographic
techniques and then recommended for the therapeutic uses.
250 ETHNOBOTANY & TAXONOMY OF ANGIOSPERMS

FLOW CHART SHOWING THE EXTRACTION PROCEDURE OF PYRROLIZIDINE ALKALOIDS

PLANT MATERIAL (50g)

PREFERABALLY FRESH
IN POWERED FORM

EXTRACT WITH ALCOHOL IN A SOXHLET

EXTRACTOR FOR 18 HOURS AT CONST.
TEMP. 40 to 60C

PLANT DEBRIES DISCARDED ALCOHOL EXTRACT

[METHYL ALXOHOL]

CONCENTRATED UNDER
REDUCED PRESSURE

ADD DILUTED AQUIOUS ACID

(2N HCl) STIRR IT FOR FEW MINUTES
ADD CHLOROFORM OR DICHLOROMETHANE

SEPARATE THE LAYERS WITH

SEPARATION FUNNEL

CHLOROFORM LAYER DISCARDED AQUIOUS ACID LAYER

[CHLOROPHYLL, LIPIDS ETC.]

FILTER BASIFY WITH CONC. AMMONIA

ADD CHLOROFORM

SEPARATE THE LAYERS WITH

SEPARATION FUNNEL

CHLOROFORM LAYER AQUIOUS LAYER

[DRIED EXTRACT CONTAINS (DISCARDED)
CRUDE ALKALOID MIXTURE
(PHYRROLIZIDINE ALKALOID)]
DETECTION OF PHYRROLIZIDINE……………..………………………FEW MEDICINAL PLANTS 251

Reagents used for the Phyrrolizidine (Alkaloids) Tests:

1. Wagner’s Reagent: 1.2g Iodine and 2g of Potassium iodide were mixed well with 5
ml of distilled water. Then the volume was made up to 100ml with distilled water.

2. Mayer’s Reagent: 1.3g Mercuric chloride and 5g Potassium iodide were mixed
separately with 60 ml of distilled water and 10 ml of distilled water respectively. Both were
individually mixed thoroughly. Then both together were mixed and volume was made up to100ml
with distilled water.

3. Dragendorff Reagent: 100g tartartic acid was dissolved in 400ml distilled water. 8.5g
basic Bismuth nitrate was added and solution was shaken for hrs. 200ml of 40% Potassium
iodide was then added and solution was shaken vigorously. After allowed to stand for 24 hrs.
solution was filtered.

Diluted Dragendorff Reagent: 100g Dragendorff Reagent was added to a solution of

100ml Tartaric acid in 500ml distilled water.

Table –1: Showing the particulars of Species Studied

Sl. Name of the Species and Place of Voucher Parts Primary Alkaloid Test Pyrrolizidine Alkaloid Test
No their family Collection Number Used Mayer’s Wagner’s Dragendoff Dagendroff “EA” “EB” hRf
Test Test Test Reagent Reagent Reagent Values
01 Aegle marmelos Corr., Byrasandra 2968 Leaf    29
(Rutaceae) 96
     
02 Azadirachta indica A. Juss. Lalbagh, 2029 Leaf    77
(Meliaceae) Bangalore    83
  
03 Cassia tora L. Jayanagar, 0937 Stem,       13
(Caesalpiniaceae) Bangalore Leaf, 99
     
Root,       93
Fruit &       68
Seed       55
   68
   75
99
  
04 Catharanthus roseus (L.) G. NMKRV 0248 Leaf    40
Don. College, 60
     
(Apocyanaceae)
Bangalore    69
   91
   99
05 Eugenia jambolana Lam. Lalbagh, 2215 Bark       Nil
(Myrtaceae) Bangalore
06 Phyllanthus emblica L. Lalbagh, 0809 Fruit
(Euphorbiaceae) Bangalore 92
     
07 Phyllanthus fraternus Webst. Lalbagh, 0845 Stem1       Nil
(Euphorbiaceae) Bangalore 0846 Stem2 Nil
     
Kanakapur
08 Tinospora cordifolia Hiers. 2046 Stem    17
(Menispermaceae) Sangama    98
  
EA=Erlich’s reagent ‘A’, EB= Erlich’s reagent ‘B’, hRf= Hundred x Relative front, + = Positive, ++ = More Positive - = Negative
 252 ETHNOBOTANY & TAXONOMY OF ANGIOSPERMS

Reference:
David, J. Robins, (1993): Pyrrolizidine Alkaloids. In: Methods in Plant Biochemistry,
Vol. 8, D.M. Day (Eds.), Academic Press, London.

Gamble, J. S. (1935): Flora of the Presidency of Madras. Vol. I to III; Botanical Survey
of India, Calcutta.

Hooker, J.D.(1878): The Flora of British India. Vol. I to VII; Reeve & Co. Ltd. London.

Mattocks, A.R. (1986): Chemistry and Toxicology of Pyrrolizidine Alkaloids. Academic

Press, London.

Mattocks, A.R. and Jukes, R. (1987); J. Nat. Prod. 50, 161-166.

Muneier, R. (1953): Bull. Soc. Chim. Biol. 35, 1225.
Ramaswami, S.V. & Razi, B. A. (1973): Flora of Bangalore District. Prasaranga,
University of Mysore, Mysore.

Saldnaha, C.J.(1984,1996): Flora of Karnataka,Vol. I&II, BH Publications, Delhi, India.

Sarath,N.Arseculeratne, Leslie Guntilaka, A.A. and Ralph,G.Panabokke, (1981):Journal of
Ethnopharmacology, 4, 159-177.
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