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CHAPTER 3

ISOLATION AND

PURIFICATION OF LUPEOL

FROM ALOE VERA

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3.1: INTRODUCTION

Although synthetic chemistry and high-throughput screening have become

popular in the last decades of the 20th century, natural products and in

particular plant kingdom are still the most valuable resources for drug

development and thus play a dominant role in pharmaceutical industries.

(Fabricant D.S. and Farnsworth N.R.(2001)) and (Balunas M.J. and Kinghorn

A.D.(2005)). Plants, for years have been important source of new drugs, new

drug leads and new chemical entities. Analysis done by Newman and Cragg

show that more than two thirds of all new chemical entities introduced

between 1981 and 2010 have some relationship to natural sources and only

30% are of purely synthetic origin (Newman D.J. and Cragg G.M. (2012)).

Through the history of combating the disease of cancer, natural products have

played an important role in the development of contemporary cancer

chemotherapy. Several plant derived compounds are being successfully

employed in cancer treatment. Among those clinically useful drugs include

paclitaxel (Taxol) ( Kinghorn, A. D and Balandrin, M. F(1993)),vincristine

(Oncovin) (Gerzon, K. et.al (1980)), podophyllotoxin(a natural product

precursor) (Jardine, I et.al(1980))and camptothecin (a natural product

precursor for water-soluble derivatives) (Wall M. E et.al(1993)). There are

many classes of plant-derived cytotoxic natural products studied for further

improvement and development of drugs.

Aloe vera also known as Aloe barbadensis is a hardy, perennial, tropical,

drought-resistant, succulent plant which belongs to the Liliaceae family and

historically has been used for a variety of medicinal purposes. It has a vast

traditional role in indigenous system of medicine like ayurveda, siddha, unani

and homeopathy. The chemical constituents of Aloe vera majorly includes ten

chemical groups i.e. amino acids, anthraquinones, enzymes, minerals,

vitamins, lignins, monosaccharide, polysaccharides, salicylic acid, saponins,

and sterols (Farooqi and Sreeramu (2001)). These includes flavanoids,

terpenoids, lectins (Vogler BK and Ernst E.(1999); King GK et.al(1995)), fatty

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acids, cholesterols, mono and polysaccharides like pectins, acemannan and

mannose derivatives (Dagne E et.al (2000), Uma R. et.al (2011)), tannins,

sterols like lupeol, β sitosterol along with vitamins, enzymes (catalase,

amylase) saponins, minerals, salicylic acid (Sumbul S. et.al (2004)), aloin,

anthrone, aloe emodin.

The heterogenous composition of the Aloe vera pulp may contribute to the

diverse pharmacological and therapeutic activities which have been observed

for Aloe vera products (Talmadge, J et.al (2004)).

The bio active compounds of aloe vera are used as anti-diabetic, anti-cancer,

anti-inflammatory, anti-bacterial and anti-septic. There use as astringent,

haemostatic, antioxidant and anti-ulcer are also known. They are effective in

treating stomach ailments, gastrointestinal problems, skin diseases,

constipation, radiation injury, wound healing, burns, dysentery, and diarrhoea

and in the treatment of skin diseases.

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Table2.1: Chemical composition and properties of Aloe vera

Chemical

group

Constituents Properties

and activity

Amino

acids

Provides 20 of the 22

required amino acids and

7 of the 8 essential ones.

Basic building blocks

of proteins in the body

and muscle tissues.

Enzymes

Anthranol, barbaloin,

chrysophanic acid,

ethereal oil, ester of

cinnamonic acid,

isobarbaloin, resistannol

Antifungal & antiviral

activity but toxic at

high concentrations

Anthraquinones Provides aloe emodin,

aloetic acid, alovin,

anthracine.

Analgesic,

Antibacterial

Steroids

Cholesterol, lupeol,

campesterol, sistosterol

Anti-inflammatory

agents, lupeol has

anticancer, antiseptic

and analgesic

properties.

Hormones Auxins and gibberellins Wound healing and

anti-inflammatory.

Salicyclic

acid

Aspirin like

compounds Analgesic

Saponins Glycosides Cleansing &

antiseptic

Minerals

Calcium, chromium,

copper, iron, manganese,

potassium, sodium and

zinc

Essential for

good health.

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In our study, we have isolated and purified lupeol from aloe vera which would

be further conjugated to the dendrimer-like poly(ethylene oxide) based

polymers to develop dendrimer based formulations and explore their

anticancer potential.

3.2: MATERIALS AND METHODS

3.2.1: Chemicals and instrumentation

All chemicals including solvents were of analytical grade. Standard of lupeol

was from sigma aldrich (Steinheim, Germany). Diethyl ether, hexane, acetic

anhydride, concentrated sulphuric acid, ethanol were obtained from

SRL(Mumbai, India). Trifluro acetic acid(TFA), methanol, acetonitrile (HPLC

grade) was obtained from Fluka( Seelze, Germany). Silica gel (0.063-.200mm)

from Qualigens fine chemicals (Mumbai, India) was used for column

chromatography. Thin layer chromatographic silica gel 60 F254 (5cm x 10cm)

plates from Merck (Darmstadt, Germany) were used for thin-layer

chromatography. HPLC , rotary evaporator

Sugars

Monosaccharides:

Glucose and Fructose

Polysaccharides:

Glucomannans/polymann

ose

Anti-viral, immune

modulating activity of

Acemannan

Vitamins

A, B, C, E,

choline, B12,

folic acid

Antioxidant

(A,C,E),

neutralises

free radicals

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3.2.2: METHODS

3.2.2.1: Plant Material

Leafs of aloe vera barbadensis miller was collected from Theni district, Tamil

nadu. A specimen has been deposited at VIT University, Vellore, Tamil Nadu,

India.

3.2.2.2: Preparation of crude extract:

Whole leaves i.e. epidermis along with gel (52g dry weight equivalent) was

extracted by maceration with diethyl ether. The maceration process was done

at room temperature for four days. After filtration, the aqueous layer was

separated from the organic layer. The collected organic layer was concentrated

under reduced pressure to obtain the crude product (2.85g, 5.48 %).

Figure 3.1: Schematic representation of the preparation of crude diethyl ether

extract of aloe vera for the isolation of lupeol.

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3.2.2.3: Qualitative analysis of crude extract

Thin-layer chromatography

The qualitative analysis of the eluted fractions for the presence of lupeol was

done using TLC. TLC was performed on pre-coated 5cm x 10cm silica gel 60

F254 plates. The chromatographic plates were developed using 10ml of

developing solvent, hexane: diethyl ether (40:60) at room temperature in a

saturated chamber by the ascending technique. Developing distance was 8 cm

for all the plates. Libermann’s bhuchard reagent was prepared by adding 1ml

of concentrated sulphuric acid and 1ml of acetic anhydride to 10ml of cold

ethanol. After developing and drying the plates were dipped in the

Libermann’s bhuchard reagent and then heated at 120ºC for 5-10 minutes and

Rf was calculated. Lupeol standard was developed in same conditions as a

reference.

3.2.2.4: Two step purification of lupeol from crude extract

Silica gel column chromatography

The crude extract of aloe vera was dissolved in diethyl ether. The suspension

was added to silica gel and evaporated to dryness. The residue was placed on

top of the silica gel (60-120 mesh) column packed using 100% hexane. The

gradient elution was done using hexane: diethyl ether (100:0, 25ml x 10

fractions; 85:15, 25ml x 30 fractions; 80:20, 25ml x 10 fractions) as solvent

system. The fraction elutes were evaporated to dryness and analyzed for the

presence of lupeol using thin layer chromatography (TLC).

Final purification of lupeol using reverse phase HPLC

Final purification of lupeol from aloe vera was performed on a RP C18 column

(4.6mm X 75mm) at room temperature using methanol: acetonitrile

(containing 0.1% TFA) as solvent system. The purification was performed by

following isocratic elution with 30:70 v/v of methanol: acetonitrile (containing

0.1% TFA) and flow rate of 0.8ml/min and run time of 15 minutes. The peak

corresponding to lupeol was collected and evaporated to dryness. Lupeol

standard as a reference was subjected to same conditions. Percentage yield of

purified lupeol: 13.9%

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Figure 3.2: Schematic representation of the two step purification of diethyl ether

crude extract of aloe vera.

Step 1: Solvent system used: hexane: diethyl ether (40:60), Developing agent

used: Libermann’s Bhuchard reagent then heated at 120ºC for 5 min.

Step 2: Column packing: 100% hexane, Solvent system used: hexane: diethyl

ether, Gradient elution: 100:0, 85:15, 80:20 (hexane: diethyl ether)

Step 3: Column used: C-18 (4.6 X 75mm), Solvent system used: Methanol:

acetonitrile (containing 0.1% TFA) (30:70) ,Isocratic elution, Flow

rate: 0.8ml/min, Injection volume:20.00 µL.

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3.3: RESULTS AND DISCUSSION

The preliminary analysis to qualitatively analyze the presence of lupeol in the

crude extract was performed using TLC on pre-coated silica gel 60 F254 plates.

By using several developing solvents the TLC method was applied to choose

the one which ensured the best separation of the different molecules in the

crude extract with special preference to lupeol. A standard lupeol solution was

used as reference. Hexane: diethyl ether (40:60) as the solvent system proved

to be the best as this resulted in the best separation of the components present

in the crude extract (fig. 3.3). In the subsequent purification steps this system

was used to check the presence of lupeol in the fractions taken. Part of the

analysis was to choose the proper developing. Several developing reagents

were tested: vanillin-sulphuric acid, iodine vapors, libermann buchard reagent.

The latter one showed the greatest selectivity that is the intensity of the color

obtained considerably exceeded that of the first two; therefore this reagent was

chosen to be a developer. The lupeol spot appeared on the TLC plate when

heated at 120º C for 5-10 min. In figure 4, the lupeol standard in lane 2

appeared violet in color with a RF value of 0.35. The crude diethyl ether extract

of aloe vera showed an intense band of lupeol at the same RF value as that of

the standard.

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Figure 3.3: Preliminary identification of lupeol using thin layer chromatography.

Lane A: crude diethyl extract of aloe vera, Lane B: Lupeol standard (purchased

from sigma Aldrich)

Conditions Used: Solvent system used: hexane: diethyl ether (40:60), Developing

agent used: Libermann’s Bhuchard reagent, heated at 120ºC for 5-10 minutes.

Rf : 0.35

A B

Rf : 0.35

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Because lupeol lack chromophores, the sensitivity of UV detection is limited

and dependent on the mobile phase. (Mitija M. et.al (2007)). Methanol:

acetonitrile (containing 0.1% TFA) (30:70) as mobile phase in Isocratic elution

with a flow rate of 0.8 ml/min enabled the separation of lupeol from the other

component and its sensitive detection at 210 nm. The lupeol standard at the

same condition showed a retention time of 7.91 min (fig. 3.4). Fig. 3.5 shows

the RP-HPLC chromatogram of the crude diethyl ether extract wherein due to

the complexity of the extract numbers of peaks are observed and no separation

could be achieved. A very minute peak at a retention time of 7.93 min was

observed which may be due to lupeol.

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Figure 3.4: C-18 RP-HPLC chromatograms of lupeol standard purchased from

sigma.

Figure 3.5: C-18 RP-HPLC chromatograms of the Crude diethyl ether extract

of aloe vera

Conditions used: Column used: C-18 (4.6 X 75mm), Solvent system used:

Methanol: acetonitrile (containing 0.1% TFA) (30:70) ,Isocratic elution, Flow

rate: 0.8ml/min, Injection volume:20.00 µL.

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A two step purification process which involved a combination of silica gel

chromatography followed by HPLC was performed to obtain purified lupeol.

The first step involved the purification by silica gel chromatography. This step

helped in reducing the complexity of the crude and improved the separation

efficiency in HPLC. A gradient elution was performed and based on TLC of

each fraction, the lupeol rich fractions were pooled and concentrated (fig 3.6).

As shown in the fig. 3.6, the silica gel chromatography was successful in

separation of lupeol along with an unknown molecule from the other

components of the crude extract. For the final purification of lupeol these

fractions were pooled, concentrated and then subjected to RP-HPLC. Fig. 3.7

shows the RP-HPLC chromatogram of the pooled fractions of silica gel

chromatography. The chromatogram clearly shows two peaks with a

prominent peak of lupeol at 7.86 minutes. Pure lupeol (396 mg, 13.9% yield)

was obtained by repeating the separation and collecting the peak

corresponding to lupeol.

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Figure 3.6: First step purification of lupeol by silica gel column chromatography

from diethyl ether crude extract of Aloe vera

Lane A: Lupeol standard (purchased from sigma Aldrich), Lane B-H: Lupeol

rich fractions (eluted with 80:20 (hexane: diethyl ether) after first step

purification of crude extract using Silica gel column.

Conditions used: Column packing: 100% hexane, Solvent system used: hexane:

diethyl ether, gradient elution: 100:0, 85:15, 80:20 (hexane: diethyl ether)

R f: 0.34

A B H

H

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Figure 3.7: C-18 RP-HPLC chromatograms of fractions after first step

purification of the crude extract using silica gel chromatography.

Conditions used: Column used: C-18 (4.6 X 75mm), Solvent system used:

Methanol: acetonitrile (containing 0.1% TFA) (30:70) ,Isocratic elution, Flow

rate: 0.8ml/min, Injection volume:20.00 µL

The purified lupeol would be further conjugated to the dendrimers and this

drug formulation would be studied for its cytotoxic activity against cancer cell

lines.