The document describes a procedure to isolate mentha oil from mentha leaves using hydrodistillation with a Clavenger apparatus. It involves extracting the volatile oils from the leaves through distillation with water in the apparatus. Volatile oils are complex mixtures found in plants that are odorous and evaporate at room temperature. They are identified and standardized based on their volatile oil content and characteristics. The procedure aims to isolate mentha oil and identify menthol within it using thin layer chromatography.

1. Pharmacognosy and Phytochemistry-II (13PH0503)
FoP, Marwadi University, Rajkot | B. Pharm. Semester-V (2021-2022) Page |38
Experiment No.: 11
AIM: To isolate sennosides from senna leaves.
REQUIREMENTS:
Apparatus: Porcelain dish, Conical flask, Beaker, Measuring cylinder, Funnel, Stirrer, Glass
plate.
Reagents:
Methanol / Ethanol, Silica gel, Acetic acid, Senna leaves, Benzene, HNO3, Calcium chloride,
Potassium hydroxide, Sodium sulphate.
THEORY:
It is obtained from the dried leaflets of Cassia angustifolia, Cassia acutifolia belonging to
family Leguminoceae (Casalphenaceae). Sennosides are the anthraquinone glycosides.
Senna glycoside, also known as sennoside, is a medication used to treat constipation and
empty the large intestine before surgery. The medication is taken by mouth or via the
rectum. It typically begins working in minutes when given by rectum and within twelve
hours when given by mouth. It is a weaker laxative than castor oil. Molecular formula is
C42H38O20.
PROCEDURE:
The weighted quantity of dried powdered Senna leaves was extracted with benzene for
30 min on electric shaker, filtered in vacuum and benzene get distilled off.
The left-over marc was dried at room temperature and extracted with 70% methanol
for three hrs. and filtered under vacuum.
The marc was re-extracted with 50 ml of 70% methanol for 15min, filtered and the
methanolic extracts get combined.
The methanolic extract was concentrated to 1/4th volume, acidified to pH 3.2 by adding
HNO3 with constant stirring.
It was set aside for 15 min. at 50C, filtered.
1 g of anhydrous calcium chloride in 10 ml of denaturated spirit was added with
constant stirring.
The pH of the solution was adjusted to 8 by addition of potassium hydroxide (KOH) and
set aside for 15 min., filter and collect the precipitate obtained in as calcium sennoside
dried and weighed.
The percentage yield was also calculated.
IDENTIFICATION BY CHEMICAL TESTS:
Borntager's Test:
To 3 ml sample, dilute sulphuric acid (H2SO4) was added, boiled and filtered. To the cold

2. Pharmacognosy and Phytochemistry-II (13PH0503)
FoP, Marwadi University, Rajkot | B. Pharm. Semester-V (2021-2022) Page |39
filtrate, equal volume of benzene was added and shaken. The organic solvent was
separated and ammonia was added. It was observed that the ammonical layer turned pink
confirming the presence of anthraquinone glycoside presence.
Modified borntager's Test:
To 5 ml sample, 5 ml ferric chloride and 5ml dilute hydrochloric acid was added. It was
then heated for 5 min in boiling water bath. cooled and benzene was added. It was shaken
well, separated and equal volume of dilute ammonia was added. It was observed that the
ammoniacal layer turned pink confirming the presence of anthraquinone glycoside.
IDENTIFICATION BY TLC:
Sennoside is identified by thin layer chromatography.
Stationary phase: Silica gel GF
Solvent System: n-propanol: ethyl acetate: water: glacial acetic acid (40:40:29:1)
Spraying reagent: HNO3 - KOH reagent.
After drying the TLC plate, Sennosides, is detected at ___ Rf value.
RESULT:
1. The percentage yield of total sennosides from senna leaves was found to be ______.
2. The Rf value of sennosides from senna leaves was found to be _____.
REFERENCES:
1. Practical Pharmacognosy by K. R. Khandelwal
2. Practical Pharmacognosy by S. Joshi and V. Aeri
________________

3. Pharmacognosy and Phytochemistry-II (13PH0503)
FoP, Marwadi University, Rajkot | B. Pharm. Semester-V (2021-2022) Page |40
Experiment No.: 12
AIM: To isolate mentha oil from mentha leaves by hydro-distillation method using
Clavenger's appatatus and identify menthol by TLC method.
REQUIREMENTS:
Apparatus: Beaker, Stirrer, Measuring Cylinder, Clavenger's apparatus.
Reagents: Water, Mentha leaf.
THEORY:
The volatile oils are complex mixtures of odorous principles found in various plant species.
They called volatile because they evaporate at room temperature, but they are also
referred to as ethereal oils or essential oils. They are mixtures of from ten to hundreds of
components.
Common properties of volatile oils:
They have a characteristic, usually pleasant odour, and they are usually colorless or pale
yellow, except for chamomile, absinth and yarrow oil (blue or greenish-blue). Their density
is usually less than 1, except for clove, cinnamon and mustard oil. During their long-term
storage, volatile oils may oxidize and resinify, which may result in a change or odour. To
prevent this, they should be kept in a dry environment, in tightly-stoppered, Fall (not partly
empty), dark glass containers. Volatile oils are almost immiscible with water but they have
a slight solubility in it, which is enough to form aromatic waters. On the other hand, they
are completely soluble in ether, alcohol and most organic solvents.
Determination of volatile oil content:
The clavenger apparatus consists of the following Parts (See Fig.). The apparatus described
below is recommended but any similar apparatus may be used provided that it permits
complete distillation of the volatile oil. All glass parts of the apparatus should be made of
good quality resistance glass. Pharmaceutical significance of aromatic drugs is due to their
odorous principal that is volatile oils such crude drugs are standardized on the basis of
their volatile contain.