1

CHAPTER I
1. INTRODUCTION

Plants are the important sources of inexpensive drugs for human and animals
throughout the whole world and called herbal medicine. In Myanmar one of the effective
medicinal plants is Aloe vera Linn, (Sha-zaung-Letpat). The aloe vera leaves contains
“gel” tissue (poly saccharide rich inner leaf mesophyll), provides a reservoir of water and
has been attribute multiple bioactive properties and digestive health. 25 % of aloe vera
plants are used for medicine and 10 aloe vera species are traded commercially . Over the
last centuries, the plant aloe vera have been extensively studies for various therapeutic
activities, such as anti-bacterial, anti-viral, anti-cancer activities as well as
immunoregulative and hepatoprotective properties . This research priority studies the
phytochemical, physicochemical and elemental analysis of aloe vera plants.

1.1 Botanical Aspects

Kingdom - Plantae
Family - Aloaceae
Genus - Aloe
Botanical name - Aloe vera L. Burm. f.
English name - Aloe vera
Myanmar name - Sha-Zaung-Letpat
Identification - Department of Botany (MUB)
Part used - Leaves

Aloe vera
 2

(a)

(b) (c)

Figure 1.1 Aloe vera (a) plant (b) leaf (c) cross section of leaf

1.2 General Descriptions of Aloe vera

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Aloe vera species has been used in folk medicine for over 2000 years and has
used as an important component in the traditional medicine of many countries . Aloe vera
is a perennial plant of the Xanthorrhoeaceae family and also placed by most sources in the
Liliaceae family. Aloe vera is a succulent leaves and the medicinal and cosmetic properties
of the gel obtained from them. There is over 400 different species well known, and aloe
barbadensis, aloe arborescens and aloe chinensis are the most popular. The leaf
parenchyma (aloe gel) is colourless and tasteless, and has been used in the treatment of
skin care. The gel obtains from aloe vera leaves contain primarily of water and
polysaccharides such as pectin, cellulose, hemicellulose, glucomannan, acemannan and
mannose derivate. The plant aloe vera has beneficial effects to humans and uses in the
formulation of food product due to its therapeutic an functional properties .

1.2.1 Medicinal uses of Aloe vera

The clear gel from the aloe plants is rubbed on the skin as an ointment to treat
wounds and burns. Historically, Native American use aloe vera for stomach disorder and
intestinal disorder including constipations, hemorrhoids, colitis and clone problems. Aloe
vera serves as a natural cleaner, powerful in penetrating tissues, relieving pain associated
with joints and muscles bactericidal, a strong antibiotic, virucidal when in direct contact
with long periods, fungicidal, anti-inflammatory, breaking and digesting dead tissues and
moisturizing tissues (Lawrence, et al., 2009). The first gained popularity of aloe vera plant
was reported of successful use of freshly cut leaves in treating X-ray burns. In recent
times, the oral consumption of aloe vera has been promoted.

1.2.2 Varieties uses of Aloe vera

Three types of extracts from aloe vera were gel extract, whole leaf extract and
decolorized whole leaf extract, and the remaining fourth types of commercial material is a
dried latex, which has been used as laxative. The green part of the leaf can be made into a
juice or dried and taken orally as a laxative. The plant, aloe vera is used in many
commercial products in various forms as beverages, capsules, powders and as a flavoring.
Food products include health and soft drinks, yogurts, jams, instant tea granules, candies,
 4

alcoholic beverages and ice creams were produced from aloe vera extract. Aloe vera
decolorized whole leaf extract are popular as dietary supplements for various systemic
aliments. The anthraquinone components of these products appear to very significantly in
their content of aloe emodin and aloin, the major anthraquinone constituent of aloe vera
latex (Elsohly, et al., 2007).
Aloe vera plant is used as a hair styling gel especially curly and fuzzy hair. It is
also used for production of makeup, moisturizers, soaps, sunscreens, shampoos and
lotions. Aloe vera gel is useful for dry skin conditions, especially eczema around the eyes
and sensitive facial skin (Rajeswari, et al., 2012).

1.3 Chemical Constituents of Aloe vera Linn

“Acemannan” (molecular formula C66H100NO49-) extracted from aloe vera has
various medicinal properties like osteogenic, anti-inflammatory and anti-bacterial. Also,
acemannan is known to have anti-viral and anti-tumar activities in vivo though activation
of immune responses. So aloe vera has immense potential as therapeutic agent (Sierra-
Garcia, 2014). Acemannan, a polysaccharide extracted from aloe vera gel could affect
bone formation (Nejatzadeh-Barandozi, 2013).

Figure 1.2 Structure of acemannan

According to the data report, aloe vera contains 75 potentially active
constituents such as vitamin, enzymes, minerals, sugars, lignins, saponins, salicylic acid
 5

and amino acid. Aloe vera contains a lot of vitamin group as vitamins A (beta-carotene),
vitamin C and E, which serves as antioxidants, vitamin B12, folic acid and choline. Aloe
vera contains 8 enzymes: alias, alkaline phosphatase, amylase, bradykinase,
carboxypeptidase, catalase, cellulase, lipase, and peroxidase. Bradykinase helps to reduce
excessive inflammation when applied to the skin topically, while others help in the
breakdown of sugars and fats. It also provides minerals such as calcium, chromium,
copper, selenium, magnesium, manganese, potassium, sodium and zinc. Aloe vera also
contain sugars as monosaccharides (glucose and fructose) and polysaccharides
(glucomannans [beta-(1,4)-acetylated mannan]/ polymannose). Aloe vera provides 12
anthraquinones, which are phenolic compounds and traditionally known as laxatives.
Aloin and emodin act as analgesics, anti-bacterials and anti-virals. Aloe vera contains 4
plant steroids as fatty acids, cholesterol, campesterol, b-sisosterol and lupeol. All of these
have anti-inflammatory action and lupeol also possesses antiseptic and analgesics
properties. 20 of the 22 human required amino acids and 7 of 8 essential amino acids also
contain in aloe vera plants .
 6

Figure 1.3 Structure of aloe emodin

Figure 1.4 Structure of aloin

1.4 Aim and Objectives

Aim
To determine phytochemical constituents, soluble matter contents,
qualitative and quantitative elemental analysis, and nutrient values of Aloe vera
Linn. (Sha-Zaung-Letpat) leaves.
 7

Objective
To collect the plant sample (Sha- Zaung- Letpat) and identify the sample.
To separate the soluble matter contents in the leaf sample.
To investigate the phytochemical constituents in the sample.
To determine the elemental contents by EDXRF.
To examine the nutritional values by AOAC methods.
To determine the Ca content in aloe vera by using AAS.

CHAPTER II
2. MATERIALS AND METHODS
2.1 Collection of Plant Sample
Aloe vera Linn. (Sha-Zaung-Letpat) was collected from Ayeyarwady Region.
The leaves of the plant sample was cleaned and washed with distilled water. And then,
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they were cut into small parts and air dried at room temperature. They were powdered by
grinding machine. The dried powdered samples were stored in a desiccator.

2.2 Determination of Soluble Matter Contents

The soluble matter contents of Sha-Zaung-Letpat leaves were determined by
dissolving in some organic solvents such as ethanol, methanol, pet-ether, acetone,
chloroform, ethyl acetate as well as in water.

2.2.1 Determination of ethanol soluble matter content

Air dried sample powder (1 g) was weighed and placed in a conical flask. 95 %
ethanol (25 cm3) was added and the flask was stoppered with a cork. The flask was shaken
continuously for 24 hours and the suspension was allowed to stand for 24 hours. The
contents were filtered rapidly through a filter paper (Whatmann No.1 paper) and washed
with small portion of ethanol to ensure complete removal of ethanol soluble matter. A
portion of the filtrate was taken in a beaker and evaporated to dryness on a water bath and
dried at 100 C to constant weight.
The difference in the weight of the beaker before and after the experiment was
taken as the ethanol soluble matter content.
At least three times of experiments were repeated as the similar procedure
mentioned above and the average of the ethanol soluble matter content is described in
Table (3.1).

2.2.2 Determination of methanol soluble matter content

Methanol soluble matter content of Sha-Zaung-Letpat leaf sample was
determined by the method, solvent and protocol was described in section (2.2.1) by using
methanol instead of ethanol.
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The experiment was repeated three times. The average methanol soluble matter
content is shown in Table (3.1).

2.2.3 Determination of pet-ether soluble matter content

Pet-ether soluble matter content of Sha-Zaung-Letpat leaf sample was determined
by the method given in section (2.2.1) by using pet-ether instead of ethanol.
The experiment was repeated three times. The average pet-ether soluble matter
content is shown in Table (3.1).

2.2.4 Determination of acetone soluble matter content

Acetone soluble matter content was determined by the method given in section
(2.2.1) by using acetone instead of ethanol.
The experiment was repeated three times. The average acetone soluble matter
content is shown in Table (3.1).

2.2.5 Determination of water soluble matter content

Water soluble matter content was determined by the method given in section
(2.2.1) by using water instead of ethanol.
The experiment was repeated three times. The average water soluble matter
content is shown in Table (3.1).

2.2.6 Determination of chloroform soluble matter content

Chloroform soluble matter content was determined by the method given in
section (2.2.1) by using chloroform instead of ethanol.
The experiment was repeated three times. The average chloroform soluble
matter content is shown in Table (3.1).
2.2.7 Determination of ethyl acetate soluble matter content
Ethyl Acetate soluble matter content was determined by the method given in
section (2.2.1) by using ethyl acetate instead of ethanol.
 10

The experiment was repeated three times. The average ethyl acetate soluble
matter content is shown in Table (3.1)
All extracts were stored in fridge-freezer for further experiments.

2.3 Qualitative Phytochemical Analysis

Following standard protocols were used for qualitative analysis of samples to
check for the presence of Alkaloids, -Amino acids, Carbohydrates, Flavonoids,
Glycosides, Phenolic compounds, Proteins, Reducing sugars, Steroids, Saponins, Tannins
and Terpenoids.

2.3.1 Test for alkaloids

2 mL of each extracts were boiled with 1% hydrochloric acid (15 mL) for about
3 minutes and filtered. The filtrate was divided into two portions and tested with
Dragendroff’s reagent and Mayer’s reagents. Appearance of dark orange or purple color
indicates the presence of alkaloids .

2.3.2 Test for -amino acids

2 mL of each extract was boiled with distilled water (25 mL) for about 10
minutes and filtered. An aliquot portion of the filtrate was transferred to a filter paper with
the help of a micropipette and allowed to dry. Then filter paper was sprayed with
ninhydrin reagent. After spraying, the paper is heated for 10 min at 105 °C when most
amino acids give purple or greyblue colours; proline (and hydroxyproline) are distinctive
in giving yellow colours .

2.3.3 Test for carbohydrates

2 mL of each extract was boiled with distilled water (10 mL) for about 20
minutes and filtered. A few drops of freshly prepared 10% - naphthol was added to the
filtrates placed in test tube. This test tube was shaken and inclined at an angle of 45.
Concentrated sulphuric acid (1 mL) was slowly put along the side of test tube. Observation
was made to see if a red ring formed between the two layers .
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2.3.4 Test for flavonoids

2 mL of each extract was added with few drops of 20% sodium hydroxide,
formation of intense yellow colour is observed. To this, few drops of 70% dilute
hydrochloric acid were added and yellow colour was disappeared. Formation and
disappearance of yellow colour indicates the presence of flavonoids in the sample extract .

2.3.5 Test for glycosides

2 mL of each extract was boiled with distilled water for about 10 minutes and
filtered. The filtrate was treated with 10% lead acetate solution. The formation of white
precipitate indicates the presence of glycosides .

2.3.6 Test for phenolic compounds

2 mL of each extract was boiled with distilled water (10 mL) for about 20
minutes and filtered. The filtrate was then treated with a few drops of 5% iron (III)
chloride solution or three drops of freshly prepared (1:1) mixture of 1% potassium
hexacyanoferrate (III) and 1% iron (III) chloride solution. Blue or green colour solutions
indicate the presence of phenols .

2.3.7 Test for proteins

To 2 mL of each extract, 1 mL of 40 % sodium hydroxide and few drops of 1%
copper sulphate were added; formation of violet colour indicates the presence of peptide
linkage molecules in the sample extract .

2.3.8 Test for reducing sugars

2 mL of each extract was boiled with 5 N sulphuric acid about 10 minutes and
filtrated. The filtrate was then neutralized with dilute 5 N sodium hydroxide solutions and
the resulting solution was boiled with Benedict’s solution for about 2 minutes.
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Observation was made to see if yellow or brick-red precipitates come down when the
solution was allowed to cool .

2.3.9 Test for steroids

2 mL of each extract was dissolved in 20 mL of pet-ether and filtrated. The
solution was added with acetic anhydride (2 mL) followed by concentrated sulphuric acid
(1 mL). Observation was made to see if solution turned to blue colour or blue green colour
indicating the presence of steroids .

2.3.10 Test for saponins

2 mL of each extract was introduced to a test tube followed by the addition of
distilled water and the mixture was vigorously shaken for a few minutes. Observation was
made to see if frothing took place .

2.3.11 Test for tannins

10% of alcoholic ferric chloride was added to the 2 mL of each extract;
formation of brownish blue or black colour indicates the presence of tannins .

2.3.12 Test for terpenoids

2 mL of each extract was added to (20 mL) of chloroform for 30 minutes and
filtered. The filtrate was evaporated to dryness in vacuum and the residue was dissolved in
ethanol (2 mL). The solution was transferred to a watch glass and the solvent was
evaporated to dryness as a water bath. Then, the residue was dissolved in acetic anhydride,
using a glass rod. The solution was treated with a drop of concentrated sulphuric acid.
Observation was made to see brick-red colour if the terpenoids were present .

2.4 Quantitative Phytochemical Analysis

2.4.1 Determination of total alkaloid content in plant sample
Chemicals
 13

Acetic acid, ethanol, concentrated ammonium hydroxide, dilute ammonium

hydroxide.
Procedure
2 g of the sample was weighed into a 250 mL beaker and 200 mL of 10 %
acetic acid in ethanol was added and covered and allow to stand for 4 hour. This was
filtered and the extract was concentrated on a water bath to one-quarter of the original
volume. Concentrated ammonium hydroxide was added dropwise to the extract until the
precipitation was complete. The whole solution was allowed to settle and the precipitated
was collected and washed with dilute ammonium hydroxide and then filtered. The residue
is the alkaloid which was dried and weighed , . The total alkaloid content of aloe vera leaf
was describe in section 3.3.1.

2.4.2 Determination of total carbohydrate content in plant sample

Apparatus
Cary win 60 UV/ visible spectrophotometer
Chemicals
Ethanol, methanol, water, glucose, 5 % phenol, concentrated sulphuric acid.
Procedure
Quantitative estimation of total carbohydrate present in plant sample was
examined by using phenol sulphuric acid method. Phenol-sulphuric acid method is the
most reliable and easiest method among the quantitative assay for carbohydrate
estimation. This method is widely used to determine the total concentration of
carbohydrate present in food. The results are expressed in the terms of a single
carbohydrate, usually glucose. A 0.2,0.4, and 0.6 mL of working standard (with 0.1 mg/mL
conc.) glucose was taken in boiling tubes and the final volumes of each tube was made 1
mL by adding distilled water. 1 mL of 5 % phenol and 5 mL of concentrated sulphuric acid
was added one by one in each tube and shook well so that the phenol and sulphuric acid
get mixed thoroughly with working standard. After 10 minutes all the tubes were placed in
water bath at 25-30 C for 15 minutes. Blank was set with 1 mL of distilled water. Then
the whole process following phenol and sulphuric acid method was repeated 0.2 mL of
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different extract aloe vera sample . Then measure the color at 490 nm . The absorbents of
standard glucose and all extract of aloe vera were shown in Table 3.3, 3.4 and Figure 3.2.

2.5 Determination of the Physicochemical Properties of Aloe vera

2.5.1 Determination of water content
Water content from (Aloe vera Linn.) samples were determined by oven-drying
method.
Apparatus
Porcelain crucibles, electric oven, desiccator
Procedure
The dry sample (1 g) was placed in a pre-weighed porcelain crucible and kept
in air for 2 hours. It was kept in an oven at 105 C for 1 hour. This was cooled in a
desiccator and then weighed again. The process of heating, cooling and weighing was
repeated until a constant weight was achieved . The amount of water in the sample was the
calculated in Table (3.5).

2.5.2 Determination of total ash content

The ash % (s) was determined by the ashing method.
Apparatus
Porcelain crucibles, a hot plate, a desiccator, and an electric furnace
Procedure of Pre-ashing
The sample (1 g) was placed in a preweighed porcelain crucible. The sample
was carefully heated to a temperature of less than 200 C to remove water and prevent the
sample from catching fire. This preliminary heating was done till the sample was
thoroughly charred.
Procedure of Ashing
The charred sample was placed in from the door of the muffle furnace.
Incineration was done at 550 C for 1 hour, a white ash was obtained and this was
removed from the furnace. Then the crucible was cooled in a desiccator and weighed. The
 15

process of heating, cooling and weighing was repeated until a constant weight was
obtained . The total ash value of the sample was then calculated in Table (3.5).

2.5.3 Determination of protein contents

The protein content was determined by AOAC Official Method.
Chemicals
Anhydrous potassium sulphate, sodium hydroxide (pellets), sulphuric acid
(sp.gr.1.84, 98 %w/v) and hydrochloric acid (sp. gr. 1.18, 3.6 % w/v).
Preparation of standard 0.1 M hydrochloric acid solution
Hydrochloric acid (8.27 mL) was slowly added to 20 mL of distilled water and
the volume made up to 100 mL to obtain a 1 M solution. This 1 M solution (100 mL) was
diluted with distilled water and the volume up to 1 L in a volumetric flask.
Preparation of standard 0.1 M sodium hydroxide solution
Sodium hydroxide (4.0 g) was dissolved in distilled water and the volume
made up to 1L with distilled water.
Methyl red solution
Methyl red (0.1 g) was dissolved in 60 mL of 95 % ethanol and volume made
up to the mark in a 100 mL volumetric flask.
Apparatus
A micro-kjeldahl flask, a digestion unit, a distilling flask, 250 mL beakers,
50 mL burette, and a water condenser

Procedure
The sample (5.0 g) was introduced into a dried Pyrex Kjeldahl flask. The
catalyst mixture (7 g anhydrous potassium sulphate and 0.2 g copper (II) sulphate) and
concentrated sulphuric acid (12 mL) were then added. The flask was partially closed by
means of a funnel and the contents were digested by heating the flask in an inclined
position on the digester. The mixture was heated gently for about 30 minutes and heating
was continued vigorously for about 2 hours until the solution become clear.
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Then the flask was allowed to cool and 75 mL of distilled water were added.
The diluted solution was transferred to a distilling bulb and allowed to cool for less than
25 °C and added sodium hydroxide solution were added to make contents strongly
alkaline. The contents were distilled by direct heating. The liberated ammonia was allowed
to absorb with the tip of condenser immersed in 50 mL of 0.1 M standard hydrochloric
acid and 5 drops of methyl red in the receiver until ammonia has distilled (150 mL
delicate). After removing the receiver, the tip of condenser was washed with distilled
water. The ammonia distillate was titrated with 0.1 M standard sodium hydroxide solution
until the colour changed from pink to colourlelss. A blank determination was also carried
out as described above but distilled water was used in the place of the sample solution.
The amount of total protein present in the sample was calculated . The protein content of
the sample was calculated in Table 3.5.

2.5.4 Determination of crude fiber

Crude fiber is the loss on ignition of dried residue remaining after digestion of
the sample with (1.25 %) sulphuric acid and (1.25 %) sodium hydroxide solution under
specific conditions.
Chemicals
Sodium hydroxide pellets, sulphuric acid (sp.gr.184, 98 % w/v)
Apparatus
round-bottomed flask (500 mL), a Buchner funnel, and volumetric flasks

Procedure
The air-dried sample (2.0 g) was extracted with petroleum ether by stirring,
settling and decanting for three times. The extracted sample was air-dried and transferred
to a round-bottomed flask. Then 30 mL of 0.12575 M sulphuric acid was added to the
sample followed by 170 mL of warmed sulphuric acid. The solution was then refluxed for
about 30 minutes and filtered through a Buchner funnel . The insoluble matter was washed
with boiling water until the find washing was free from acid in (Table 3.5).
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2.5.5 Determination of fat content

The fat content was determined by the Soxhlet extraction method.
Chemicals
Petroleum ether (bp 40-60 °C)
Apparatus
A Soxhlet extractor, a glass funnel, thimbles, round-bottomed flask (50 mL and
500 mL) a rotary evaporator, a water condenser, a 250 mL conical flask, a water bath, a
vacuum pump and a desiccator.
Procedure
The sample (5.0 g) was weighed, placed in a thimble and the contents was
placed in the central siphon portion of the Soxhlet extractor the extractor until some of it
over flowed into the flask. The petroleum ether was heated by means of a water bath. The
extraction was assumed to be completed when a small amount of extract placed on a watch
glass did not leave any residue on evaporation of solvent. Duration of about 16 hours was
required for complete extraction. The petroleum ether was removed by simple distillation
until the volume of the ether was reduced to about 15 mL. The ether solution was
transferred to preweighted beaker and the residual petroleum ether was removed and
evaporated to dryness in a water bath and reweighed again . This procedure was repeated
until a constant weight was obtained and the fat content was calculated in (Table 3.5).

2.5.6 Determination of carbohydrate content

The carbohydrates present in foods include, starch (glycogen in animal tissue),
dextrin, mono and disaccharides. The total carbohydrates content of any food can be
obtained as the difference between 100 and the sum of the percentages of moisture,
protein, fat, ash and fiber. Although individual carbohydrates can, if necessary, be
estimated separately by chemical methods, the ‘total’ carbohydrate content of a food
obtained by calculation as described above is sufficiently accurate for practical nutrition
work . The carbohydrate content of the plant sample was given in (Table 3.5).
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2.6 Determination of Relative Composition of Some Elements by Energy

Dispersive X-ray Fluorescence (EDXRF) Spectrometry
Sample
Dried Sha-Zaung-Letpat leaves sample.
Chemicals
Liquid nitrogen
Apparatus
Energy dispersive X-ray fluorescence (EDX-8000) spectrometer was
determined at the Department of Chemistry, West Yangon University, Yangon.

Procedure
The sample was placed in the sample chamber of EDX-8000 spectrometer that
can measure the 12 samples at a time. The chamber was pumped up to vacuum. Rhodium
target was used in EDX-8000 spectrometer. Each sample was run for a counting time of
about 100 seconds and the spectrum obtained was stored and analyzed in PC based multi-
channel analyzer using EDX-8000 software.

2.7 Determination of Trace Contents of Elements by Atomic Absorption

Spectrophotometer
Sample preparation
0.5 g of the dried powder sample was added to the 9 mL of freshly prepared
acid mixture (65 % HNO3 and 37 % of HCl (1:3)) and the mixture was boiled gently over
a water bath (90 C) for 4-5 hours (or until the sample had completely dissolved). During
the digestion procedures, the inner walls of the beakers were washed with 2 mL of
deionized water to prevent the loss of the sample, the last part of the digestion process, the
sample was filtered with Whatmann FP30/0.2 CA-S filter (0.2 m particle retention).
Then, a sufficient amount of deionized water was added to make the final volume up to
100 mL) .
 19

Apparatus
Beakers (150 and 250 mL), volumetric flasks (50 and 100 mL), a (10 mL)
graduated pipette, a glass funnel, a desiccator, water bath and atomic absorption
spectrophometer (AA 7000 series) at the Maubin University.
Procedure
The above solution was transferred to a flask and trace calcium content was
determined by Atomic Absorption Spectrophotometer.

CHAPTER III
3. RESULTS AND DISCUSSION
3.1 Soluble Matter Contents
Various solvent were used for extraction because successful determination of
biologically active compounds from plant material is largely dependent on the types of
solvent used in the extraction procedure. Water is universal solvent, used to extract plant
products with antimicrobial activity. Acetone was used for hydrophilic and lipophilic
component extraction of plant sample and it was volatile and has a low toxicity to the
bioassay used, it is very useful for extracting. In this study, flavonoids, tannins, phenols,
alkaloids, glycosides, -amino acids was found in acetone extract. Ethanol and Methanol
was used as a solvent to extract higher amount of poly phenol and more efficient in cell
 20

walls degradation. Methanol is more polar than ethanol but due to its cytotoxic nature, it is
unsuitable for extraction of medicinal purpose. Terpenoids have been obtained by
extractions of chloroform and methanol but very active in chloroform. Pet-ether is
commonly used for the extraction of fats and oils.
The solubility content of Aloe vera Leaf sample was determined by the
procedure described in section (2.2) using different solvents such as ethanol, methanol,
pet-ether, acetone, chloroform, ethyl acetate and water and yielded 10.34 %, 5.23 %,
0.01 %, 1.38 %, 1.48%, 1 % and 14.9 % respectively. The results are shown in Table (3.1).
According to these results, H2O soluble matter content is higher than those of
other solvents.

Table 3.1 Different Soluble Matter Contents of the Sample

No Solvents Used Content (%)

1. Ethanol 10.34
2. Methanol 5.23
3. Pet-ether 0.01
4. Acetone 1.38
5. Chloroform 1.48
6. Ethyl acetate 1.00
7. Water 14.9
 21

Figure 3.1 Soluble matter contents in Aloe vera Linn. (Sha-Zaung-Letpat) leaf
sample

3.2 Preliminary Phytochemical Analysis of Aloe vera Linn.

The leaves of Aloe vera Linn. sample were carried out by phytochemical
examinations. It was found that alkaloids were found in water, ethanol, methanol and
acetone extract because most of the alkaloids were soluble in ethanol and polar solvents,
-amino acids was found in all extract. The presence of carbohydrates, flavonoids,
glycosides, phenolic compounds, proteins, reducing sugars, steroids, saponins, tannins and
terpenoids was found in aloe vera leaf sample. The detail resulting data are shown in Table
(3.2).
 22

Table 3.2 Results of Preliminary Phytochemical Analysis of Aloe vera Linn. (Sha -
Zaung-Letpat) Leaf Sample

Types of Solvents
No. Test reagents Observation
compound
H2O EtOH MeOH PE EA CHCl3 Acetone
Dragendorff’s
reagent Orange ppt + + + - - - +
1. Alkaloids
Mayer’s White ppt + + + - - - +
reagent
2. α-Amino acids Ninhydrin Purple spot + + + + + + +
10 % α-
3. Carbohydrates naphthol, conc; Red ring + + + - - - -
H2SO4
 23

20 % NaOH
4. Flavonoids and 70 % Pink colour + + + - - - -
H2SO4
10 % lead
5. Glycosides White ppt + + + - - - +
acetate
Phenolic 5 % FeCl3
6. Blue colour + + + - - - +
compounds solution
40 % NaOH, 1
7. Proteins Violet colour - + + - - - -
% CuSO4
Reducing Benedict’s
8. Brick red + + + - - - -
sugars solution
Acetic
9. Steroids anhydride, Green colour - - - - - + -
conc; H2SO4
10. Saponins Distilled water Frothing + + + + + + +
10 % ferric Brownish
11. Tannins + + + - + - +
chloride blue
Reddish
12. Terpenoids conc; H2SO4 - + + - - - -
brown

(+) = present, (-) = absent.

3.3 Quantitative Phytochemical Analysis
3.3.1 Determination of total alkaloids content in plant sample
Total alkaloids content was determined by , and the detail procedure was
shown in section 2.4.1. The residual alkaloids was dried and weighted, 0.3031 % of total
alkaloids were found in plant sample.

3.3.2 Determination of total carbohydrates content in plant sample

Quantitative estimation of total carbohydrates present in plant sample was
calculated by the following table.
 24

Table 3.3 Absorbance at 490 nm with different concentration of working standard of

glucose solution
Blank 1 2 3
Glucose solution (mL) 0 0.2 0.4 0.6
Distilled water (mL) 1 0.8 0.6 0.4
5 % phenol solution (mL) 1 1 1 1
Concentrated sulphuric acid (mL) 5 5 5 5
Absorbent (nm) 0 0.1237 0.2324 0.3648
Concentration (mg/mL) 0 0.02 0.04 0.06
 25

Figure 3.2 Standard calibration curve of standard glucose solution at 490 nm

Table3.4 Absorbance at 490 nm with different extract of sample solution

Ethanol Methanol Watery

extract extract extract
Sample solution 0.2 0.2 0.2

Distilled water (mL) 0.8 0.8 0.8

5 % phenol solution (mL) 1 1 1
Concentrated sulphuric acid (mL) 5 5 5
Absorbent (nm) 1.6411 7.2906 1.5176
 26

Concentration (mg/mL) 0.2730 1.2130 0.2525

3.4 Determination of Physicochemical Properties of Aloe vera

The determination of percentage of the moisture, ash, protein, crude fiber,
crude fat, carbohydrate and energy values of dried powder sample, Sha-Zaung-Letpat leaf
sample were carried out according to the reported method. These results of nutritional
values are shown in Table (3.5). It is well-known that the higher moisture content, the
greater changes for the growth of micro-organisms, but if the moisture content is lower
than 12 percent, the changes for the growth of micro-organisms are greatly minimized.

Table 3.5 Results of Physicochemical Properties of Aloe vera Linn. (Sha-Zaung-

Letpat)
Sr. Test Test Method Result (%)
 27

No. Parameter
1. Water content Oven drying method 81.00
2. Ash Ashing method 15.00
3. Protein Macro-Kjeldahl distillation 0.31
4. Crude Fiber Digestion method 1.27
5. Crude fat Soxhlet extraction 0.16
6. Carbohydrate Substraction method 2.26
7. Energy value calculation method 11.72
(kcal / 100 g)

Figure 3.3 Physicochemical properties of Aloe vera Linn. (Sha-Zaung-Letpat)

 28

3.5 Elemental Analysis by Energy Dispersive X-ray Fluorescence (EDXRF)

Spectrometry
X-ray spectrometer permits simultaneously analysis of light element to heavy
element. Shimadzu EDX-8000 spectrometer can analyze the elements from Na to U under
vacuum condition.
In this work, relative abundance of elements present in Sha-Zaung-Letpat leaf
sample was determined by EDXRF spectrometer. The EDXRF spectrum of Sha-Zaung-
Letpat leaf sample is shown in Figure (3.4) and the data are described in Table (3.6). It can
be found that K, Ca, and Si were found in Sha-Zaung-Letpat leaf sample as major
elements. Trace metals contain in Aloe vera leave were examined by EDXRF
spectroscopic method, these method obtained by relative abundance of elements in
sample. Pb and As were not detected in the leaf sample by EDXRF, indicating that there is
no toxic effect for the consumers of aloe vera.
 29

Figure 3.4 EDXRF spectrum of Sha-Zaung-Letpat leaf sample

Table 3.6 Relative Abundance of Some Elements in Aloe vera Linn Sample by using
EDXRF Spectrometry

No. Elements Relative Abundance (%)

1 Potassium 4.760
2 Calcium 3.784
3 Silicon 0.844
4 Phosphorous 0.389
5 Sulphur 0.228
6 Iron 0.110
7 Manganese 0.044
 30

Figure 3.5 Relative abundance (%) of elements in Sha-Zaung-Letpat leaf by using

EDXRF method

3.6 Determination of Ca Content by Atomic Absorption Spectroscopy

The major element Ca content was determined by AAS spectroscopy in and

8.3623 ppm of Ca was found in 0.5 g of aloe vera sample.
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CHAPTER IV
4. CONCLUSTION
From the determination of soluble matter contents, ethanol (10.34 %),
methanol (5.23 %), petroleum-ether (0.01 %), acetone (1.38 %), chloroform (1.48 %),
ethyl acetate (1.00 %) and water (14.9 %) were found to be present. The yield percent of
soluble matter contents may be important and standardization of herbal drugs of medicinal
practice.
Some qualitative phytochemical constituents such as alkaloids, -amino
acids, carbohydrates, flavonoids, glycosides, phenolic compounds, proteins, reducing
sugars, steroids, tannins, terpenoids, and saponins were observed in Sha-Zaung-Letpat leaf
sample. The phytochemical results indicate to be useful for pharmacological study and
effective to the human health.
For quantitative phytochemical constituent such as 0.3031 % of total alkaloids
and total carbohydrates were determined in three extract such as ethanol contain 0.2730
mg/mL, methanol 1.2130 mg/mL and water 0.2525 mg/mL were found in selected sample.
The contents of water (81.00 %), ash (15.00 %), protein (0.31 %), crude fiber
(1.27 %), crude fat (0.16 %), carbohydrate (2.26 %) and calories (11.72 kcal/ 100 g) were
determined by AOAC methods. From these results, Aloe gives the good supplemental
nutrients to human.
Semi-quantitative determination of trace elements in the Sha-Zaung-Letpat
leaves was determined by ED-XRF technique. It was found that the Sha-Zaung-Letpat
leaves contained K (4.76 %), Ca (3.75 %) and Si (0.84 %) as main constituents.
Furthermore, the elemental analyses of Sha-Zaung-Letpat leaves was detected
by atomic absorption spectrophotometer. The result showed that 8.3623 ppm of Ca was
present as main constituent.
 32

5. SUGGESTION FOR FURTHER WORK

Quantitative phytoconstituents should be performed in this plant.

Moreover, bioactive compounds should be isolated from Aloe vera plant and the
compounds obtained should be identified by modern instrumental methods.
 33

REFERENCES