ISOLATION AND CHARACTERIZATION OF INDOLE

ALKALOIDS FROM Rauwolfia serpentine

ABSTRACT:

This research work was carried out by M. Pratyusha Reddy, P. Hari

Chandana and K. Harsha Vardhan Reddy in the Natural Products Laboratory of Indian Institute
of Science and Technology.

Rauwolfia.serpentina is an evergreen tropical plant, very important in the

pharmacopoeia of Ayurvedic medicine for the past 4000 years, effective against various central
nervous system and intestinal disorders. In1957, 1500 papers on the use of snakeroot for
schizophrenia and other mental illness were published which revolutionized treatment of mental
illness in replacing heretofore used treatments such as electric shock therapy and lobotomies.
Several research papers were published on the alkaloids present in fresh samples of the plant but
their degradation with time in long standing roots of R. serpentina was not heretofore studied and
hence constitutes the aim of this work.

In this work we have extracted the alkaloids in the old air dried roots of R.
serpentina by acid-base extraction methods and the alkaloids were isolated by chromatographic
techniques-preparative TLC and column chromatography. The isolated alkaloids were then
analysed and characterisezed by Nuclear Magnetic Resonance, Mass Spectroscopy and Infra Red
Spectroscopy.

From our work we could successfully isolate and characterize the indole
alkaloid, yohimbine along with phthalate esters.

KEY WORDS: Rouwolfia serpentine , Alkaloids , NMR spectroscopy , IR Spectroscopy ,

Yohimbine , Pthalate esters
1. INTRODUCTION:

1.1.0. Natural Products:

Natural products are chemical compounds or substances produced in living systems. They often
have a biological activity which may be useful in pharmaceuticals. These natural chemical
compounds may be extracted from tissues of terrestrial plants, marine organisms or
microorganism fermentation broths. A crude extract from any one of these sources typically
contains novel, structurally diverse chemical compounds.

Most biologically active natural product compounds are secondary metabolites that are not
directly involved in the normal growth, development or reproduction of organisms. The function
and importance of secondary metabolites to the organism is usually of an ecological nature as
they are used as defenses against predators, parasites and diseases. Hence the products are mostly
used for medical purposes. Approximately 63% of all approved small molecule drugs are derived
from natural products, or are nature-inspired semi synthetic derivatives of natural products.

1.2.0. Natural Products from the plant kingdom:

Plant kingdom is the major sorce of natural products. Plant kingdom has been an important
source for a wide variety of chemicals such as pharmaceuticals, food additives and
agrochemicals. Mankind as well as members of the animal kingdom depend on plants either
directly or indirectly for their living. Almost every part of the plant is useful and can be exploited
for natural products. The % of the alkaloid depends on the geographical place from where the
plant is collected and also the season of collection. Generally samples from Assam have a higher
% of alkaloids (2.57 %) than the other parts of India and December is the best month for the
collection for getting more % of alkaloids.

However, age of the plant has no effect on the % of alkaloid content (up to 4 years). Ajmalicine,
ajmaline, isoajmaline, ajmalinine, chandrine, rauwolfinine, renoxidine, rescin-namine,
reserpiline, reserpin, reserpinine, sarpagine, serpentine, serpentinine, tetraphyllicine, yohimbine,
3-epi-a-yohimbine are the minor alkaloids identified from samples collected from India.
 SERPENTINE

SARPAGINE

AJMALICINE

The chemical composition of some of the alkaloids present in Rauwolfia serpentina are:

Name: Formula:

Ajmalicine C20H26N2O2

Rauwolfine C20H26N2O3

Rauwolfinine C29H26N2O2

Reserpine C33H40N2O9
Serpentinine C21H20N2O3

Tetraphylline C22H26N2O4

RESERPINE

1.3.0.Rauwolfia serpentina:

1.3.1.Scientific classification:

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Gentianales

Family: Apocynaceae

Genus: Rauwolfia

Species: R. serpentina

The Apocynaceae or dogbane family is a family of flowering plants that includes trees, shrubs,
herbs, and lianas.Many species are tall trees found in tropical rainforests, and most are from the
tropics and subtropics, but some grow in tropical dry, xeric environments.The family, as
currently recognized, includes some 1500 species divided in about 424 genera. My plant of
interest belongs to the genus Rauwolfiaand species serpentina.

2. MATERIALS AND METHODOLOGY:

2.0.1. PLANT MATERIAL: Plant material has been identified and collected by Dr. B. Rama rao

METHODOLOGY:

2.1.0. Methanol Extraction:

 I have got 500 grams-dry weight of plant material (roots of R. serpentina air dried over a
period of 5 years).The voucher specimens of the samples have been kept in the
laboratory.
 The experimental sample was taken (500gms), which were the long air dried roots of
Rauwolfia serpentina and were crumbled and pulverized into fine powder.
 The air dried and powdered plant material of same weight was extracted with the known
volume (3*50ml) of methanol for 5 days at room temperature.
 This was kept for a week with constant stirring at regular intervals of time. All the
organic compounds extracted into methanol were then separated using filter paper and
suction filter.
 Left over solid residue was again soaked in methanol solvent for better extraction.
 The steps were followed for 3-4 times for the complete transfer of pant extracts from the
plant roots into the solvent.
 The extract was then concentrated in a rotavapour (suction of solvent vapors by
maintaining vacuum or under reduced pressure) at 40 ̊C.

3.2.0. Acid Base Extraction:

Acid-base extraction is a procedure of using sequential liquid-liquid extractions to purify acids

and bases from mixtures based on their chemical properties. This procedure is routinely used for
the isolation of natural products especially alkaloids.
The fundamental theory behind this technique is that salts, which are ionic, tend to be water-
soluble while neutral molecules tend not to be.
The addition of an acid to a mixture of an organic acid and base result in the acid remaining
uncharged, while the base is protonated. If the organic acid, such as a carboxylic acid, is
sufficiently strong, its self-ionization can be suppressed by the added acid. Conversely, the
addition of a base to a mixture of an organic acid and base will result in the base remaining
uncharged, while the acid is deprotonated to give the corresponding salt. Once again, the self-
ionization of a strong base is suppressed by the added base.

2.2.1.Acidification and Defatting :

 The concentrated extract was acidified to a Ph of 4.0 by 1N HCl.

 The solution was poured in a glass jar or separatory funnel and 25%-50% of the solution
volume of xylene was added to the solution.
 The neck of the jar was plugged and was then gently turned over 50 to 100 times as
vigorous shaking result in emulsification of the solution, which would be difficult to
separate.
 Once mixed, the jar was allowed to sit and separate out into the different layers.
 When this was done, 3 layers were observed-- a top layer of solvent, a middle fatty layer,
and a bottom layer containing the acidic aqueous solution (and the alkaloids).
 Using a separatory funnel, the bottom aqueous layer was drained out and the top layers
were thrown away.
 The process was iterated several times till all the lipids were transferred into xylene
leaving just alkaloids in the bottom layer.

Three layers on acidification Lipids on defatting

.3.2.2.Basification :

 After defatting, Sodium Hydroxide was slowly added until ph is around 10 (Dry NaOH is
not added since the reaction is exothermic).
 Once basification was done further work was continued immediately, as alkaloids left in
strongly basic solution break down
 Making the solution basic turns the alkaloids into their free base forms, which are soluble
in xylene.

2.2.3.Extraction Of Free Bases:

 Once the solution is basic, xylene was added using the same ratio as used during the
defatting process. Again, the solution was mixed thoroughly but gently to avoid
emulsification of solution.
 The solution was allowed to sit till it separates out into two layers. The bottom layer
contains a basic aqueous solution, and on the top was a xylene layer which contains the
alkaloids. Using a separatory funnel or a siphoning process, the xylene layer was
collected and set aside.
 Since there will still be significant alkaloids in the aqueous layer, this process was
repeated two more times. All the xylene was then combined and the remaining aqueous
solution was discarded.
 To this xylene solution 1N HCl of same ratio was added in a separating funnel. The
organic compounds here alkaloids are then transferred into acidic solution. The step was
repeated by taking 1N HCl again so that all the alkaloids were completely transferred to
the acidic layer. The presence of alkaloids was verified by using thin layer
chromatography.
 The basification of solution was carried out using Sodium Carbonate(Na 2CO3) . The
solution ph was maintained around 7-8 but not less then 7 .The presence of alkaloids was
verified by thin layer chromatography.
 The compound was next extracted into ethyl acetate by using a separating funnel. The
step was repeated until all the alkaloids were transferred to ethyl acetate and then was
concentrated in rotavapour at 40 ̊C.
 The concentrated plant material was then chromatographed on TLC for finding out the
extact mobile phase for clear separation that is to find out the degree of mobility or Rf
(retardation factor) of a particular molecule or compound which mainly depends on its
polarity.

2.3.0. Thin Layer Chromatography:

TLC (Thin Layer Chromatography) is the separation technique in which sample is tested for the
different constituents in it by running it on a thin silica layer (which acts as a stationary phase)
spread over a glass plate, with the help of a solvent system (which acts as a mobile phase).
If the compound is more polar, the more will be its mobility and less will be its Rf value. This
trial plate technique though is time taking and strenuous, has helped us to go directly for
PREPARATIVE TLC and COLUMN CHROMATOGRAPHY. Not only that but to go for long
or short column i.e., if the Rf values of the compounds are too close we have to go for long
column and if they are far we have to go for a short ones.
The mobility of the compounds can be assayed in three ways:
1. If the compound shows chromatographic effect, this can be possible only for the
compounds which show conjugated (alternate) double bonds or lone pair of electrons
under transition. Then the compound can be observed and identified under U.V radiation.
2. When exposed to Iodine vapours.
3. When sprayed with 10% methanolic sulphuric acid solution or with ansaldehyde. For
further clarification, the sprayed plates were kept for charring on hot plate

2.3.1. Trial TLCs:

Several trials were made on trial TLC plates by increasing the polarity of the solvent
systems, to find out the exact mobile phase.
i. With Hexane.
ii. With ethyl acetate and hexane.
iii. With methanol and hexane.
iv. With acetone and hexane.
v. With chloroform and hexane.
 Among the various above trials, fifth solvent system showed a crude separation. For
further clear separation, another round of trials were made by increasing or decreasing
polarity i.e. by changing the ratio of the solvents in the solvent system:
a) Hexane:chloroform in 10:1.
b) Hexane:chloroform in 9:1
c) Hexane:chloroform in 8:2
d) Hexane:chloroform in 7:3
e) Hexane:chloroform in 6:4
f) Hexane:chloroform in 5:5
g) Hexane:chloroform in 4:6
h) Hexane:chloroform in 3:7
 The solvent system (f) showed clear separation on TLC plate.
 Solvent system(f) Solvent system in U.v light

2.3.2. Preparative TLC:

 With this idea the material was then chromatographed very carefully over preparative
thin layer chromatography(20cm*20cm).
 The solvent system used for chromatography was chloroform and hexane in 1:1 ratio,
conducted in a closed TLC room.
 Two major bands containing organic compounds were observed and were noted as
1A(top) and 1B(lower).
 Both the bands were carefully bordered and scraped, followed by subsequent filtration
through a silica gel bed using ethyl acetate in column chromatography.

Preparative TLC after the silica in the bands is scraped for extraction

2.4.0. Spectral Studies:

 The filtrate of both the samples was gently evaporated to dryness.
 A portion of the dry mass was individually weighed and dissolved in a known volume of
CDCl3(2-3 ml) for compound determination by 1H1-NMR.Authentics were taken as
reference.
 Some of the dry mass was weighed and dissolved in methanol-chloroform solution for
compound determination by Mass Spectroscopy.
 The remaining weight of the filtrate along with regenerated sample from CDCl 3 was later
dissolved in methanol-KBr solution and sent for Infra Red Analyses.
4.1.0. Analysis of Spectral data for Compound 1A:

STRUCTURE:

One broad signal in 1H NMR is observed in the region of ð 11.94 indicating the presence of one
ene amine proton in the compound. From the IR spectra a signal is also observed at 3448.3cm -1
indicating the presence of secondary amines. In 1H NMR the C4 and C7 protons appear as
doublets in the region of ð 7.8 and C5 and C6 protons appear as muliplets in the region of ð
7.6(2H,d) indicating the presence of aromatic protons in the compound. The signals in the region
of ð 4.4 -4.2 and ð 0.9-1.9 correspond to remaining protons in the compound.

The signal from IR spectra at 1646.4cm-1 indicates the presence of ester groups.

From the Mass Spectra [M]+ peak is observed at 391.

From all the above data it can be concluded that the structure of the given compound is as follows:

The above compound is an alkaloid yohimbine.

Compound 1-b Spectral studies
4.2.0. The Analysis of Spectral Data for Compound 1B:
From the 1H NMR, signals are observed at ð7.36 and ð7.25 doublets corresponding to the protons at C 3
and C4 respectively. The signals at
ð4.04 doublet, ð2.01 multiplet and ð .01
doublet represents the protons a11, 21 , 31
and 41 respectively.

From IR spectra a signal is observed at

1743cm-1 which represents ester group
is present in the compound.

+
From Mass Spectra [M+Na] peak is
observed at 301.

From all the above data it can be

concluded that the structure of the given compound is as can be seen below:
The above compound is a phthalate ester Diisobutyl Phthalate(DIBP).

5. CONCLUSIONS
From the dissertation work carried out and from the results obtained thereafter I have observed
that by following the protocol I’ve designed, I could successfully isolate the indole alkaloid,
Yohimbine and the phthalate ester, Diisobutyl Phthalate(DIBP) from the long standing roots of
Ruuwolfia serpentina.

6. REFFERENCES:

1. H. Falkenhagen, I.N. Kuzovkina, I.E. Alterman, L.A.Nikolaeva, and J. Stockigt. Nat. Prod. Lett. 3, 107
(1993).

2. A. Petit, C. David, G.A. Dahl, J.G. Ellis, P. Guyon, F. CasseDelbart, and J. Tempe. Mol. Gen. Genet.
190, 204 (1983).

3. M. Hesse. I12 Progress in Mass Spectrometry, Indolalkaloide,Teil I. Erlirerl by H. Budzikiewicz.

Verlag Chemie, Weinheim. 1974.

4 . E. Wenkert, C.-J. Chang, H.P.S. Chawla, D.W. Cochran,W. Hagaman, J.C. King, and K. Orito. J. Am.
Chem. Soc.98, 3645 (1 976).
5. F.E. Bader, D.F. Dickel, R.A. Lucas, and E. Schlitter. Experientia, 10, 298 (1954).

6. A. Hofmann. Helv. Chim. Acta, 37, 314 (1954).

7. R. Goutarel, A. Hofmann, M.M. Janot, A. LeHir, and N.Neuss. Helv. Chim. Acta, 40, 156 (1957).

8. L. Toke, K. Honty, L. Szab6, G. Blasko, and C. Szintay. J.

V. E. Klyushnichenko, S. A. Yakimov, T. P. Tuzova, Ya. V. Syagailo, I. N. Kuzovkina, A. N. Wulfson

and A. I. Miroshnikov, Determination of indole alkaloids from R. serpentina and R. vomitoria by high-
performance liquid chromatography and high-performance thin-layer chromatography, Journal of
Chromatography A,Volume 704, Issue 2, 9 June 1995, Pages 357-362.

9. Joachim Stö ckigt, Artur Pfitzner and Joachim Firl, Indole alkaloids from cell suspension
cultures of Rauwolfia serpentina benth , Plant Cell Reports, Volume 1, Number 1 / August,
1981,Pages 36-39.
10. Heike Falkenhagen and Joachim Stockigt, Indole alkaloids from "hairy roots" of
Rauwolfiaserpentine, Can. J. Chem.

71, 2201 (1993).

11. Joseph Monachino, Rauvolfia serpentina--lts History, Botany and Medical Use,The New York
Botanical Garden.

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