See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/303824573

" PHYTOCHEMICAL PROFILING OF HABENARIA EDGEWORTHII HOOK.F. EX

COLLETT (VRIDDHI) OF DEOBAN UNDER CHAKRATA FOREST DIVISION "
Submitted by CHANDRA PRAKASH SEDAI

Thesis · June 2015

DOI: 10.13140/RG.2.1.1690.8409

CITATIONS READS

3 82,737

1 author:

Chandra Sedai
President Chure Terai Madhesh Conservation Development Board
8 PUBLICATIONS 10 CITATIONS

SEE PROFILE

All content following this page was uploaded by Chandra Sedai on 07 June 2016.

The user has requested enhancement of the downloaded file.

 DIVISIONAL ATTACHMENT ON

“PHYTOCHEMICAL PROFILING
HABENARIA OF
EDGEWORTHII HOOK.F. EX COLLETT (VRIDDHI) OF
DEOBAN UNDER CHAKRATA FOREST DIVISION”

Submitted by
CHANDRA PRAKASH SEDAI
M.Sc. Forestry (2013-2015)
Third semester
Submitted in requirement of partial fulfilment of the degree M.Sc.
Forestry

Under the guidance of

Dr. V.K. Varshney

Scientist-F & Head

Chemistry Division

FRI (DEEMED) UNIVERSITY

Indian Council of Forestry Research & Education
(An autonomous body of Ministry of Environmental & Forests, Govt. of India)
P.O. NEW FOREST, DEHRADUN- UTTARAKHAND -248006

ii
 Contents

1. Abstract ................................................................................... 2
2. Introduction ............................................................................ 3
2.1 Habenaria edgeworthii Hook.f. ex Collett (Vriddhi) .............. 3
2.2 Phytochemical Properties and therapeutic uses: ................... 5
3. Literature Review .................................................................... 7
4. Objective ................................................................................. 8
5. Study Area ............................................................................... 8
6. Materials and Methods ........................................................... 9
6.1 Plant Material ......................................................................... 9
6.2 The phytochemical profiling ............................................... 10
6.2.1 Alkaloids .......................................................................... 11
6.2.2 Glycosides ....................................................................... 12
6.2.3 Terpenoids ...................................................................... 16
6.2.4 Tannins............................................................................ 16
6.2.5 Phenolic compound ........................................................ 17
6.3 Moisture content determination ......................................... 17
7. Results and Discussion .............................................................. 18
8. References: ............................................................................... 24

iii
List of figures:-
Figure 1: Habenenaria edgeworthii a) Natural habitat of the species b) A whole
natural plant c) Flowers of the plant (Deoban field photos, 2014)………………….....…6
Figure 2: Habenenaria edgeworthii a) Herbarium b) A whole natural plant in
Deoban Nursery (Deoban field photos, 2014) …………………………………………….....6
Figure 3: Map of the study area (Map not in scale) from google maps ………........…9
Figure 4: Different Reagent used in the laboratery for chemical..………………………
Figure 5: Habeneria edgeworthii a) Dried Rhizome b) Dried rhizome crushed in a
grinder for chemical test c) Mayer’s test for Alkaloid d) Dragendroff’s test for
Alkaloid...................................................................................................................23

List of tables:-
Table 1: Chemical test for Alkaloids…………………………..………………………………………
Table 2: Chemical tests for anthraquinone glycosides……………………………………….18
Table 3: Chemical tests for steroid and triterpenoid glycosides…………………………
Table 4: Chemical tests for saponin glycosides………………………………………………….
Table 5: Chemical tests for cardiac glycosides…………………...………………………………
Table 6: Chemical tests for coumarin glycosides………………...…………………………….
Table 7: Chemical tests for flavonoid glycosides……………...…………………………………
Table 8: Chemical tests for steroid and triterpenoids…………………….....……………….
Table 9: Chemical tests for tanins………………………………………………………………..…...21
Table 10: Chemical tests for phenolic compound………………………………………………
Table 11: Moisture content estimates of the rhizome of H. edgeworthi……………21

i
 1. Abstract

"Astavarga" is used as tonic which promotes body heat, dries up serious fluids,
carminative and antitussive, and are useful in vitiated conditions of pitta and vatta, agalactia,
seminal weakness, internal and external haemorrhages, cough, bronchitis, burning sensation and
general debility having antioxidant and vitality strengthening properties.(Acharya Balkrishna,
2012) (A. Dhyania, 2010). This divisional attachment discusses the one of the rare and valuable
Astavarga plants i.e. Habenaria edgeworthii. This plant emerged as a good source of sodium
while various antioxidant assays provided evidences on the antioxidant potential of the species
and highlighted the possibilities of harnessing nutritional and antioxidant potential of these. The
samples of the plant is collected from Deoban forest which is a picturesque landscape of
Chakrata Forest Division, Uttarakhand, India with an altitude of 2200 meters to 3025 meters
above sea level. The phytochemical studies determination of Moisture content and isolation and
extraction of alkaloids of the rhizomes of Habenaria edgeworthii is performed in the laboratory
of Chemistry division, Forest Research Institute, Dehradun, India.

The observation on the phytochemical profiling of the rhizomes of Habenaria

Edgeworthii showed the presence of Alkaloids, Coumarin glycosides and Phenolic compounds.
The compounds such as Anthraquinone glycosides, Saponin glycosides, Steroids and Terpenoids
glycosides, Flavonoid glycosides, Cardiac glycosides and Tanins was confirmed to be absent in
the rhizomes of Habenaria edgeworthii. The moisture content of the rhizomes was founded to be
2.51%.
Keywords: Phytochemicals, Plant metabolites, Secondary metabolites, primary metabolites,
Alkaloids, Glycosides, Terpenoids, Volatile oils, Tannins, Resins.

2
 2. Introduction

Astavarga eight plants belong to two families, 'Liliaceae' comprising Meda (Polygonatum
verticillatum (L.) Alt.), Mahameda (Polygonatum cirrhifolium (Wall.) Royle), Kakoli (Roscoea
alpina/purpurea), Ksheerakakoli (Lilium pollyphyllum), and 'Orchidaceae' comprising Jeevak
(Malaxis acuminata), Rishibhak (M. muscifera), Riddhi (Habenaria edgeworthii), Vriddhi (H.
intermedia) is important ingredient of various classical Ayurvedic formulations like
Chavyanprasha. Ashtavarga has been assigned various medicinal properties by ancient Materia
Medica dealing with Ayurveda (Acharya Balkrishna, 2012)

All of these plants have their natural habitats in Himalayas particularly the North West
Himalaya in Jammu & Kashmir, Uttarakhand, and Himachal Pradesh between elevation of 1500
and 4000 masl. Their natural habitat are specific in ecological environment and hence these
occur only in small pockets (Acharya Balkrishna, 2012).
Ashtavarga is a subject of rigorous botanical research. Although work has been done on
identification of medicinal plants mentioned under Ashtavarga, still lot is to be done to identify
the true representatives. Taking in to consideration the medicinal properties of these medicinal
plants, phytochemical and pharmacological investigations are the need of the hour to
scientifically validate the claims. Among them Habenaria edgeworthii is of great therapeutic
value.

2.1 Habenaria edgeworthii Hook.f. Ex Collett (Vriddhi)

Sanskrit name: Vriddhi

Botanical name: Habenaria edgeworthii Hook.f. Ex Collett, Planthanthera edgeworthii (Hook.f.
ex Collett) R.K. Gupta
Family: Orchidaceae
English name: Edgeworth’s platanthera

Synonyms: Lakshmi, Mangala, Rathanga, Rishisrista, Saravajanpriya, Siddhi, Sukha, Vasu and
Yuga.
Botany: Vriddhi is orbicular in shape and its fruit is twisted on right side. Plant consists of small,
fusiform, ellipsoid to subglobose tubers giving rise to an erect, leafy stem carrying 3 to 4 lower
broadly ovate, becoming ovate to oblong-lanceolate up the stem, bract like above, sessile leaves.
Stem is somewhat flexuous leafy stem, covered with hairs. Its flower is yellowish green,
deflexed in buds, in cylindrical inflorescence., spike 20-25cm long, 3 cm abroad, dense: bract
lanceolate, equaling the ovary in length; sepals green, margin ciliolate, broadly ovate; petals

3
bright yellow, spur longer than ovary, 1.5-2.5 cm long directed upwards; staminal column 2-
3mm anther canal short, stigmatic processes- short, oblong; ovary twisted, glabrous. Fruit in
capsule form usually open laterally. Seed is numerous, dust like small seeds. Flowering occurs at
July to August and fruiting occurs in September- October. Its tuber is hoary and porous (Sharma,
2005).
Habit and Habitat: Found in the western Himalayas, Pakistan, the eastern Himalayas and Nepal
in scrub on open hillsides at elevations of 1500 to 2500 meters as a medium sized, growing in
warm to cold terrestrial region. A tuberous terrestrial orchid, growing up to 75cm in height. In
India it is found across the Himalayas in the North western parts Jammu & Kashmir, Himachal
Pradesh and uttarakhand up to an elevation of 2500- 3500 m. In Uttarakhand - Mussoorie-
Jaberkhet, Kyarphulli, Company garden, Deoban, Tehri-nagtibba, west of Dhanolti, Uttarkashi-
Jamuna valley, Kharsil, Har-ki-Dun: Chamoli- the valley of flowers, Gobindghat; Pauri- Khirsu:
Pithoraghat- Tejam jankhola valley, Kali valley; Sarju valley; Nainital, Bhowali, Ramgarh,
Fatehgarh, Above Malli tal ; Binsar, Almora, Lorakhet, Rani khet to Chaubatia (Sharma, 2005).
Parts used: Tubers
Ayurvedic dynamics: Sweet in taste and pacifies vata and pitta but aggravates kapha.
Actions: Cooling and spermopiotic.
Therapeutics: Diseases of the blood.
Phytochemistry: Tubers contains bitter substances, starch and minerals and phenolic
compounds.
Actions: Cooling and spermopiotic.
Therapeutics: It is useful in blood diseases, burning sensations, thirst, fever, cough, asthma,
muscular pain, sprains, arthritis, sciatica, insanity, leprosy, skin diseases, anorexia, worms,
emaciation, gout, hyperdypsia, , cataplepsy, helminthiasis and general debility,

4
2.2 Phytochemical Properties and therapeutic uses:

H. edgeworthii emerged as a good source of sodium. While various antioxidant assays

provided evidences on the antioxidant potential of the species and highlighted the possibilities of
harnessing nutritional and antioxidant potential of these (Rawat, et al, 2014)
Tuber has properties of cooling, emollient, brain tonic, blood purifier, appetizer, rasayana and
tonic. Tuber is also useful in burning sensation, excessive thirst, fever, cough, asthma, insanity,
leprosy, skin diseases, anorexia, worms, emaciation, gout and general debility (A. Balkrishna,
2012)

Medicinal uses:
1. Cephalic diseases
Mahamayura ghrta processes with Vriddhi and other herbs is useful in rasa- raktadi
dhatugata vikara, srota and indriya vikara, svarabhransa, ashthma, cough, facial
paralysis, vaginal diseases, blood disorders and semen related disorders.
2. Rejuvenation and vitality strengthening
Astabarga churna processed with vrddhi and other herbs is similar in properties to
Jivaniyagana churna and is used as dhatuvrddhikara, gunakara and roganasaka.
Chyavanaparasa prepared with Vrddhi and other herbs including Astavarga in
appropriate dose and in prescribed way is useful in rasayana guna (Sharma, 2005).
Dosage: Powder 2-3 gram or as advised by physician.
Status: It is a rare species because it grows in association with grasses and is being cut in early
stages before seed are mature.

5
Figure 1: Habenaria edgeworthii a) Natural habitat of the species b) A whole natural plant c)
Flowers of the plant (Deoban field photos, 2014)

Figure 2: Habenaria edgeworthii a) Herbarium b) A whole natural plant in Deoban Nursery

(Deoban field photos, 2014)

6
 3. Literature Review

Three Himalayan medicinal plants (Habenaria intermedia, H. edgeworthii, and

Roscoea procera), widely used in vitality strengthening and ayurvedic formulations in India,
from Dhanoulti district Tehri in Uttarakhand by were assessed for nutritional phytochemical
constituents, and antioxidant activity by Rawat et al, 2014. These target species emerged as a
good source of minerals and possessed important micro elements.
Individually, H. intermedia contained a high content of total phenols, thiamins,
tannins, and calcium; R. procera was rich in potassium and iron content; and H. edgeworthii
emerged as a good source of sodium. While various antioxidant assays provided evidences on
the antioxidant potential of target species, greater antioxidant potential of H. intermedia as
compared to the other two species was revealing. (Rawat et al 2014)
The observed Phytochemicals (in mg/g dry weight) in H. edgeworthii are Alkaloids (0.47 ±
0.05b), Tanins (2.24 ± 0.02a), Phenols (5.31 ± 0.06a), Flavonoids (3.05 ± 0.05a), Thiamine (5.75
± 0.06b), Riboflavin (3.56 ± 0.3a), Mineral ash (3.56 ± 0.3a), Fiber (4.61 ± 0.1c) & Fat (6.27 ±
0.8c).

The Mineral elements and its composition (mg/100 g dry weight) of rhizomes of
Habenaria Edgeworthii in Dhanaulti region were Sodium (62.90±0.01c), Pottasium
(219.27±0.04b), Calcium (158.65±0.07a), Lithium (3.39±0.01b), Copper (4.76±0.02a), Zinc
(4.51±0.03b), Iron (84.54±0.02a), Magnesium (6.69±0.06b) and Cobalt (5.37±0.12b).
Species specific responses in phenolic compounds were revealing. For example, gallic acid was
observed highest in R. procera (92.84 mg/100 g dry weight), whereas hydroxybenzoic acid was
detected only in H. intermedia (18.51 mg/100 g dry weight) and H. edgeworthii (7.56 mg/100 g
dry weight). The Phenolic compounds (mg/100 g dry weight) of Habenaria Edgeworthii rhizome
in Dhanaulti Region is Gallic acid (6.0±1.0b) and Hydroxybenzoic acid (7.6±0.04a). The values
are mean ± SD of three determinants; values with different letters in a row are significant at p <
0.01. (Source: Rawat et al, 2014)
ABTS radical scavenging assay showed significantly (p < 0.01) greater antioxidant
potential in H. intermedia (2.723 mM AAE/100 gm dry weight) followed by H. edgeworthii
(1.709 mM AAE /100g dry weight) in methanol extract. The other two antioxidant assays
followed similar trends and revealed significantly higher antioxidant activity in the case of H.
intermedia as compared to H. egdeworthii and R. procera. (Rawat et al, 2014)
There is systematic gap in phytochemical studies regarding its investigation on
phytochemical studies and antioxidant potential. Considering this gap the present study is
concerned in phytochemical profiling and isolation of alkaloids.

7
 4. Objective

The objective of this research was to conduct Phytochemical Profiling of the rhizomes of
Habenaria edgeworthii Hook.f. ex Collett (Vriddhi) of Deoban under Chakrata Forest Division
and to determine its moisture content.

5. Study Area

Deoban is a picturesque landscape of Chakrata Forest Division, Uttarakhand, India. As

the Deoban name suggest “God own Forest”, it has a true peaceful environment giving inner
calmness. It has scenic hilly terrains with an altitude of 2200 meters to 3025 meters above sea
level. The area under present study lies in the Chakrata Forest Division, Dehradun, Uttarakhand
between 30º 26: 31º 2’ Latitude and 77º 38’: 78º 4’ longitude. The forest area falls under
Himalayan moist temperate forest and Lower and upper Himalayan moist Temperate forest
respectively (Srivatsava, & Agarwal, 1978).
The area is pleasantly cool even in june: the precipitation is high and mostly in the form of
snow. The climate of the area is moist temperate receiving moderate to high snowfall. The
winter is long, severe and get repeatively snowfalls during December to February.
The vegetation of the area is typically temperate comprising of Quercus leucotrichophora, Q.
floribunda, Cedrus deodara, Picea smithiana, Pinus wallichiana, Acer caesium, Rhododendron
arboretum and Myrica esculenta. In shrubaceous and herbaceous stratum species like
Cotoneaster acuminate, Rosa macrophylla, Berberis aristata, Vibernum species, Arundinaria
spp., Daphane spp., Skimia laureolla, Anemone mercifolia, Taxacum officinale, Plantago
major and Carex spp.

8
 Figure: 3 Map of the study area (Map not in scale) from google maps

6. Materials and Methods

6.1 Plant Material

Tubers/rhizomes of the target species were collected during August 2014 from Deoban
31º 2’ Latitude and and 78º 4’ longitude district Dehradun in Uttarakhand (Indian west
Himalaya). The plant was authenticated by Dr. H. B. Nathani, Systematic Botany Branch,
Botany Division, Forst Research Institute Voucher specimens, identified after consulting the
Forest Research Institute, Dehradun. The herbarium were deposited in the herbarium of
Systematic Botany division, Forest Research Institute, Dehradun, India.
Plant constituents, as the word implies are the individual chemicals from which plants are
made. These constituents are organic in nature and synthesized in plants by the activity of
individual cells. The process by which these complex organic chemical constituents are formed,
utilizing simple substances and enzymes are known as biosynthesis.

9
 As historically, plants and their products form the basis of medicines and also in present
days. Several compounds, which are pharmaceutically and medicinally important, derived from
plant sources. However, the medicinal value of plant depends on the nature of plant constituents
present in it, which is known as active principal or active constituent. Active constituents are
those chemical substances, which are solely responsible for therapeutic activity of plant. A large
number of theories have been proposed as to why these compounds are formed in plants, it is
likely that many of them are produced as part of chemical defense system to protect the
producing organism.
The chemical constituents present in plants that do not possess any definite therapeutic
value are known as inactive constituents. As the formation of different active and inactive
constituents of plants involves various metabolic pathways, hence in general the inactive plant
constituents are termed as primary plant metabolites whereas active plant constituents are termed
as secondary plant metabolites.
Primary plant metabolites are simple molecules or polymers of simple molecules
synthesized by plants, generally do not possess therapeutic as such but essential for the life of
plants and contains high-energy bonds. These are used up for the biosynthesis of secondary
metabolites. E.g. Carbohydrates, proteins, lipids and nucleic acids.
Secondary metabolites are complex organic molecules biosynthesized from primary plant
metabolites in plant cells. Unique to plants or group of plants, generally possess therapeutic
activity, neither essential for plants life nor contains high energy bonds. These are usually stored
in vacoules. Secondary metabolites are classified as: alkaloids, glycosides, tannins, phenolic
compounds, volatile oils, terpenoids, saponins, steroids, resins and bitter principles. These are
used as medicine, food, flavors, colours, dyes, poisons and perfumes etc. It is estimated that
1/4th of prescription drugs contains at least one chemical originally identified from plants.
(Ahmed Sayeed, 2014)

6.2 The phytochemical profiling

Phytochemicals are chemical compounds that occur naturally in plants (phyto means
"plant" in Greek). Some are responsible for color and other organoleptic properties, such as the
deep purple of blueberries and the smell of garlic. The term is generally used to refer to those
chemicals that may have biological significance, for example carotenoids or flavonoids, but are
not established as essential nutrients. There may be as many as 4,000 different phytochemicals
having potential to affect diseases such as cancer, stroke or metabolic syndrome.

10
 Composition of phytochemicals (Source Wikipedia.org)

Phytochemical profiling was performed by making the test solution. The collected tubers
of Habaneria Edgeworthi’s is oven dried in a dried in a hot air oven (40◦C) and powdered using
a grinder mill. Powdered samples were stored in airtight bag till further analysis.
The Phytochemical test of rhizomes of Habenaria Edgeworthi’s was tested in the
laboratory of chemistry division, Forest Research Institute, Dehradun, India following standard
protocal. Each chemical test is repeated for three times and the most occurring result was
confirmed. The chemical test was done for presence or absence of following compounds.
 Alkaloids
 Glycosides
Anthraquinine Glycoside
Saponin Glycoside
Steroid and Triteroenoid Glycoside
Cardiac Glycoside
Coumarin Glycoside
Flavonoid Glycoside
 Terpenoids
 Phenols and
 Tannins

6.2.1 Alkaloids

Alkaloids are organic compounds of basic nature hence named alkali like or alkaloids
and have one or two nitrogen atom within nucleus or outside the nucleus. Alkaloids are usually
derived from amino acids and show prominent pharmacological action in small doses. Most
alkaloids are alkaline solids like quinidine, emetine and atropine. Some alkaloids are found in
liquid form like coniine, nicotine etc. These are colorless but some of them are colored like
berberine (yellow), betain (red). Alkaloids contain carbon, hydrogen, one or more than one
nitrogen, usually oxygen and sometimes sulphur. These are optically active laevorotatory form is
pharmacologically more active than dextrorotatory. Alkaloids are bitter in taste, available in the
form of salt, less toxic and don’t show addiction property like morphine. Free bases of alkaloids
are insoluble in water and soluble in organic solvent like chloroform, ether etc. (caffeine and
colchicine are soluble in water) whereas alkaloidal salts and quaternary alkaloids are highly

11
soluble in water but insoluble in organic solvents (lobelline HCl soluble in CHCl3, quinine
sulphate is sparingly soluble in water). Alkaloids, taken in their broadest sense, may have
nitrogen atom which is primary e.g. mascaline, secondary e.g. ephedrine, tertiary e.g. atropine
and quaternary e.g. tubocurarine.

Most alkaloid skeletons are derived from amino acids whereas some derived from other
groups of molecules also such as the steroidal alkaloids with the nitrogen from glutamine or
another N donor being added in later biosynthetic steps.
Like many secondary metabolites, plants apparently synthesize alkaloids for defensive
purposes. Nicotine and derivatives are among the earliest known and most potent insecticides.
Chemical Test for Alkaloids: The chemical test are performed from neutral or slightly acidic
solution of drug following type of chemical test given by alkaloids are-
1. Dragendorff’s Test: Drug solution + Dragen droff’s reagent (Potassium Bismuth Iodide),
the formation of orange red color shows presence of Alkaloids.
Dragen droff’s reagent consists of following compounds in respective proportions
 Bismuth Nitrate (0.85 gram)
 Glacial acetic acid (10 ml.)
 Potassium Iodide (8 gram)
 Water (10 ml)
 Sodium Nitrite or H2 So 4
 Aqueous Sodium Nitrite (10%)
 Ethanollic Sulphuric Acid (10%)
 NaNo2

The test solution was prepared by dissolving a small fraction of powder mixed with Acetic Acid
to make slightly acid solution.

2. Mayer’s Test: Drug solution + few drops of Mayer’s reagent (K2HgI4), the formation of
creamy-white precipitant shows presence of Alkaloids.

6.2.2 Glycosides

In nature glycosides are formed by interaction of nucleotide glycosides like uridine

phosphate glucose with alcohol, phenol, steroid, triterpenoid and flavonoids etc. and joined by
glycosidic linkage.

12
These are non-reducing (do not reduce Fehling solution) organic substances which on hydrolysis
yields one or more sugar molecules along with non-sugar molecules. The sugar molecule known
as glycon and non-sugar molecule termed as aglycon part. Sugars are hemiacetal and occur as
oxide rings.

Glycosides can be defined as the condensation product of hydroxyl group of aglycon and
hemiacetal hydroxyl group of sugar. The aglycon may be any compound containing at least one
hydroxyl group to which glycosidal hydroxyl group of sugar joints. Glycosides are colorless,
crystalline or amorphous solid substances (flavonoids are yellow colored whereas anthracene
glycosides are red to orange) generally, poisonous in nature. These are soluble in water and
alcohol but insoluble in ether and chloroform, optically active, usually levorotatory.

Glycosidic hydroxyl group reacts with large number of organic compounds and acid liable
i.e. the organic moiety attached at glycosidic hydroxyl group is hydrolyzed with acids whereas
others are not. The sugars present in glycoside are of two isomeric form i.e. α form and β form
but all the natural glycosides contain β-type of sugar.

α- Glucose β- Glucose
Chemical Tests of Glycosides: Glycosides are the compounds with organic molecules having
attached glucose or any mono-oligo sacchrid unit. Usually, these are crystalline or amorphous
solids; optically active, soluble in water and alcohol but insoluble in organic solvents like ether,
chloroform and benzene etc. Generally, aqueous or alcoholic extracts of crude drugs are tested
with specific reagents for presence of various types of glycosides.

B. On the basis of chemical nature of aglycon and pharmacological activity

1. Anthraquinone glycosides: These are derivative of anthraquinones, possess purgative property,

may be dihydroxy phenol (chrysophanol), trihydroxyphenol (emodine), or tetra hydroxy phenols.
Anthraquinone derivatives are often orange red coloured compounds, soluble in hot water and
alcohols. e.g Sennosides A, B, C and D.

13
 Anthraquinone
Chemical tests for anthraquinone glycosides
Modified Borntragor’s Test: To 1 gm of drug add 5 ml dilute HCl followed by 5 ml ferric
Chloride (5% w/v). Boil for 10 minutes on water bath, cool and filter, filtrate was extracted with
carbon tetrachloride or benzene and add equal volume of ammonia solution, formation of pink to
red colour due to presence of anthraquinone moiety. This is used C-type of anthraquinone
glycosides.
2. Saponin glycosides: These forms honey comb like foam when shaken with water and causes
haemolysis of blood. Saponin complex organic compounds distributed in higher plants and are
toxic to cold-blooded animals and lower organisms like earthworm and fishes. These are soluble
in alcohol but insoluble in ether and light petroleum; on hydrolysis it gives aglycone known as
sapogenin (generally steroids) and sugars.
Chemical tests for Saponin glycosides

Foam test: To 1 gm of drug add 10-20 ml of water, shake for few minutes, formation of
frothing which persists for 60-120 seconds in presence of saponins.

3. Chemical tests for steroid and triterpenoid glycosides

Salkovaski test: Alcoholic extract of drug was evaporated to dryness and extracted with
CHCl3, add conc. H2 SO4 from sidewall of test tube to the CHCl3 extract. Formation of yellow
coloured ring at the junction of two liquid, which turns red after 2 minutes, indicate the presence
of steroid moiety.
4. Chemical tests for cardiac glycosides

Keller Killiani test: To the alcoholic extract of drug equal volume of water and 0.5 ml of
strong lead acetate solution was added, shaked and filtered. Filtrate was extracted with equal
volume of chloroform. Chloroform extract was evaporated to dryness and residue was dissolved
in 3 ml of glacial acetic acid followed by addition of few drops of FeCl3 solution. The resultant
solution was transferred to a test tube containing 2 ml of conc. H2SO4. Reddish brown layer is
formed, which turns bluish green after standing due to presence of digitoxose.

14
5. Coumarin glycosides: These are aromatic compounds containing benzo-α-pyrone ring system.
The alcoholic solution of coumarins shows blue-green fluorescence on addition of alkali. Some
coumarins are containing furan ring attached at 6-7 or 7-8 position in coumarin ring and are
called as furano coumarin. These are generally used externally, in skin disorders and in sun tan
preparations because they have property to absorb UV radiation of sunlight. e. g. Aesculin from
plants of family rosaceae and scopolin; furano coumarins are generally present in family
rutaceae, umbelliferae and leguminosae like psoralen, xanthotoxin, bergapten and imperatorin.

Benzo-α-pyrone
Chemical tests for Coumarin glycosides
FeCl3 test: To the concentrated alcoholic extract of drug few drops of alcoholic FeCl3
solution was added. Formation of deep green colour, which turned yellow on addition of
conc. HNO3, indicates presence of coumarins.
6. Flavone glycosides: These are complex organic compounds containing phenyl-benzo-χ-pyrone
ring system. Flavones are present in plants in free-state or in glycosidal state (O-glycoside or C-
glycoside) with its different derivatives like flavane, flavonol, flavonone, isoflavone and
chalcones. E.g. Rutin, quercitrin, hyperoside, diosmin (buchu leaf), hesperidin (lemon and
orange peel) and vitexin (Carategus).

Benzo-χ-pyrone 2-phenyl-benzo-χ-pyrone
Chemical tests for flavonoid glycosides
a. Ammonia test: Filter paper dipped in alcoholic solution of drug was exposed to ammonia
vapor. Formation of yellow spot on filter paper.
b. Vanillin HCl test: Vanillin HCl was added to the alcoholic solution of drug, formation of pink
colour due to presence of flavonoids.

15
 6.2.2 Terpenoids

These are hydrocarbons of plant origin and their oxygenated, hydrogenated and
dehydrogenated derivatives having general formula (C5H8)n. These are building blocks of
isoprene (2-methyl-buta 1, 3 diene, C5H8) units, joined together in head to tail fashion.
Terpenoids are colorless compounds, lighter than water with boiling point 150-180 °C. These are
optically active liquids (few terpenoids are solid), insoluble in water but soluble in organic
solvents. The term terpene originates from the mixture of isomeric hydrocarbons of molecular
formula C10H16 present in turpentine oil. Now, terpenes are only limited to a class of compounds
of terpenoids that is monoterpene hydrocarbon of molecular formula (C5H8)2, whereas terpenoids
represents hydrocarbon and their oxygenated derivatives, hence it can be stated that all the
terpenes are terpenoids but not vice-versa.

Isoprene (2-methyl-buta 1, 3 diene)

Chemical tests for triterpenoid
Salkovaski test: Alcoholic extract of drug was evaporated to dryness and extracted with CHCl3,
add conc. H2 SO4 from sidewall of test tube to the CHCl3 extract. Formation of yellow colored
ring at the junction of two liquid, which turns red after 2 minutes, indicate the presence of steroid
moiety.

6.2.3 Tannins

Tannins are mixture of complex organic, non-nitrogenous substances derivatives of

polyhydroxy benzoic acid (polyphenols) with ability to precipitate proteins and having high
molecular weight (500 to > 20000). These are non-crystalizable, astringent substances, soluble in
water (forming colloidal solution), dilute alkali, alcohol, glycerol and acetone while sparingly
soluble in ethyl acetate, chloroform and other organic solvents. Aqueous solutions of tannins are
acidic in nature due to presence of free phenolic and carboxylic groups. It precipitates heavy
metals, alkaloids, glycosides and gelatin (proteins) from solutions. Tannins combined with
proteins by cross-links making raw animal skin to leather and on application to living surface it
renders proteins resistant to proteolytic enzymes (astringent property), which forms the basis of
therapeutic application of tannins.

16
Chemical tests for tannins: Tannins show specific chemical reaction like solution of tannins
precipitate gelatin, alkaloids, salt of Copper, Lead and Tin etc. and shows color reaction with
K2Cr2O7, chromic acid and iron salts.
Vanilline HCl test: Solution of test drug was mixed with few drops of vanilline HCl.
Development of pink color in presence of tannins due to conversion of phloroglucinol from
catechin.

6.2.4 Phenolic compound

Chemical tests for phenolic compound

FeCl3 test: To the concentrated alcoholic extract of drug few drops of alcoholic FeCl3 solution
was added. Formation of deep green colour.

6.3 Moisture content determination

For the determination of the moisture in the rhizome of Habenaria Edgeworthii the drugs is cut
into pieces and 2 grams is taken in each beaker. The initial weight is weighed in an electronic
balance. The sample is put in a microwave in a temperature of 103ºC ± 2º. Then the final weight
is weighed for every 2-3 hours until the weight of oven dry weight of the sample become
constant. The constant weight is taken as the final weight. Then the final weight is calculated as
follows:
Moisture content = (Initial wet weight – Final oven dry weight) * 100
Final oven dry weight

17
 7. Results and Discussion

Phytochemical profiling of the rhizome was conducted using standard protocol as given
by Ahmad S., 2007. The observation was given below:
Table 1: Chemical test for Alkaloids

Experiment Observation Results

Orange red colour was Alkaloids presence was
1. Dragendorff’s Test: obtained. indicated.
Acidified solution of
rhizome (5ml) +
Dragen droff’s
reagent (2-3 drops)
2. Mayer’s Test: Creamy-white precipitate was Alkaloids presence was
Acidified solution of formed conformed.
rhizome (5ml) + (2-3)
drops of Mayer’s
reagent (K2HgI4)

Table 2: Chemical tests for anthraquinone glycosides

Experiment Observation Results

Modified Borntragor’s Test: Pink or red color was color Anthraquinone glycosides
1 gm powdered rhizome + 5 liquid was not obtained. was absent.
ml dilute HCl + 5 ml ferric
Chloride (5% w/v)
Boiled for 10 minutes on
water bath, cool and filter.
Filtrate was extracted with
carbon tetrachloride or
benzene and add equal
volume of ammonia solution.

18
Table 3: Chemical tests for Saponin glycosides

Experiment Observation Results

Foam test: 1 gm powdered Frothing was not obtained. Saponin glycosides was
rhizome + 10-20 ml of water, absent.
shake for few minutes

Table 4: Chemical tests for steroid and triterpenoid glycosides

Experiment Observation Results

Salkovaski test: Yellow coloured ring at the Triterpenoid glycosides was
Alcoholic extract of rhizome junction of two liquid was not absent.
was evaporated to dryness formed.
and extracted with CHCl3,
Add conc. H2SO4 from
sidewall of test tube to the
CHCl3 extract.

Table 5: Chemical tests for cardiac glycosides

Experiment Observation Results

Keller Killiani test: To the alcoholic Reddish brown Cardiac glycosides was
extract of rhizome + equal volume of layer was not absent.
water formed.
And 0.5 ml of strong lead acetate
solution was added, shaked and
filtered.
Filtrate was extracted with equal
volume of chloroform. Chloroform
extract was evaporated to dryness and
residue was dissolved in 3 ml of glacial
acetic acid followed by addition of few
drops of FeCl3 solution. The resultant
solution was transferred to a test tube
containing 2 ml of conc. H2SO4.

19
Table 6: Chemical tests for Coumarin glycosides

Experiment Observation Results

FeCl3 test: To the Dark green colour was Coumarin glycosides was
concentrated alcoholic extract obtained. indicated.
of powdered rhizome few
drops of alcoholic FeCl3
solution was added.

Table 7: Chemical tests for flavonoid glycosides

Experiment Observation Results

a. Ammonia test: Filter paper Yellow spot on filter paper Flavanoid glycosides was
dipped in alcoholic solution was not obtained. absent.
of powdered rhizome was
exposed to ammonia vapor.

Vanillin HCl test: Vanillin Pink colour was not obtained. Flavanoid glycosides was
HCl was added to the absent.
alcoholic solution of
powdered rhizome

Table 8: Chemical tests for steroid and triterpenoids

Experiment Observation Results

Salkovaski test: Yellow coloured ring at the Triterpenoids was absent.
Alcoholic extract of junction of two liquid was not
powdered rhizome was formed.
evaporated to dryness and
extracted with CHCl3,
Add conc. H2SO4 from
sidewall of test tube to the
CHCl3 extract.

20
Table 9: Chemical tests for Tanins

Experiment Observation Results

Vanillin HCl test: Vanillin Pink colour was not obtained. Tanins was absent.
HCl was added to the
alcoholic solution of
powdered rhizome

Table 10: Chemical tests for phenolic compound

Experiment Observation Results

FeCl3 test: To the Dark green colour was Phenolic compounds
concentrated alcoholic extract obtained. presence was conformed.
of powdered rhizome few
drops of alcoholic FeCl3
solution was added.

Table 11: Measurement of the Moisture content in the rhizome of Habenaria Edgeworthii

Beaker 1 Beaker 2 Beaker 3

Weight of empty 53.244 48.479 51.918
Beaker (gram)
Weight of sample 2 2 2
(gram)
Weight of beaker & 55.244 50.479 53.918
sample (gm) X1
Oven dry weight of 53.903 49.163 52.676
beaker & sample
after 2 hrs (gm)
Oven dry weight of 53.901 49.160 52.672
beaker & sample
after 2 hrs (gm)
Oven dry weight of 53.901 49.160 52.672
beaker & sample
after 2 hrs (gm) X2
D = X1 - X2 1.343 1.319 1.246
Moisture content% { 2.49 % 2.68% 2.36%
(D/ X2 ) *100}
The average moisture content of the rhizomes of Habenaria edgeworthii was 2.51%.

21
 Above observation on the phytochemical profiling of the rhizomes of Habenaria
edgeworthii shows the presence of Alkaloids, Coumarin glycosides and Phenolic compounds
was conformed. The compounds such as Anthraquinone glycosides, Saponin glycosides, Steroids
and Terpenoids glycosides, Flavonoid glycosides, Cardiac glycosides and Tanins are absent in
the rhizomes of Habenaria edgeworthii. The Moisture content of the rhizomes is 2.51%. The
alkaloids extraction process and its potential antioxidant and vitality strengthening properties is
needed to be studied in future.
The information generated in this study form a scientific basis for further detailed
chemical extraction of Habenaria edgeworthii for its utilization and promotion. This is the first
report on the phytochemical profiling of the species of that area. This would enrich the existing
information base on the phytochemical studies of Himalayan medicinal plants.

22
Figure 4: Different Reagent used in the laboratey for chemical test

Figure 5: Habeneria Edgeworthii a) Dried Rhizome b) Dried rhizome crushed in a grinder for
chemical test c) Mayers test for Alkaloid d) Dragendroff’s test for Alkaloid

23
 8. References:

 Acharya Balkrishna. (2012). Secrets of Astavarga Plants (for vitality and anti-aging).
Haridwar, Uttarakhand, India: Divya Prakashan, Patanjali Yogpeet, 10- 92.

 Acharya Balkrishna, (2012). Astavarga plants – threatened medicinal herbs of North -

West Himalaya. International Journal of Medicinal and Aromatic Plants , 6 (4), 661-676.
 Agarwal, V. S., Srivatsava, V. K., & Agarwal, N. C. (1978). Geology of the Parts of
Dehradun District. Lucknow, Westbengal, India: Geographical Survey of India 5-15.

 Ahmad, S. (2007). Introduction of the Plant Constituents and their Tests. New Delhi,
India: Department of Pharmacognosy and Phytochemistry 2-40.

 Ravi kant Verma, J. V., Verma, R. K., Verma, J., & Thakur, K. (2012). Distribution
pattern, Survival threats and Conservation of Astavarga orchids in Himachal Pradesh,
North West Himalaya. 1(2), 94-102.

 Rawat, S., Andola, H., Dhyani, P., Jugram , A., Bhatt, I. D., & Rawal, R. S. (2014).
Assessment of Nutritional and Antioxidant Potential of Selected Vitality Strengthening
Himalayan Medicinal Plants. 17(3), 703- 712.

 Sharma, B. D. (2005). Vitality Strengthening Astavarga Plants. Haridwar, Uttarakhand,

India: Divya Publishers, Divya Yog Mandir (Trust) 2-48.

24
View publication stats