CERTIFICATE

This is to certify that the dissertation entitled “PHYTOCHEMICAL ANALYSIS OF

TABERNAEMONTANA DIVARICATA USING LEAF EXTRACT SCREENING OF IMPORTANT

ACTIVITY” through sequential solvent extraction .A prospective study, submitted to the K., in
partial fulfilment of the requirement for the award of the degree of Bachelor Of Science, In
Biochemistry is a record of original research Work done by during the period 2023-2024 o
under my supervision and guidance and the dissertation has not formed the basis for the
award of any Degree / Diploma / Associate /Fellowship or other similar titles to any
candidate of university.

HEAD OF THE DEPARTMENT SIGNATURE OF THE GUIDE

 DECLARATION

I Hereby declare that the dissertion entitled “PHYTOCHEMICAL ANALYSIS OF

TABERNAEMONTANA DIVARICATA USING LEAF EXTRACT AND SCREENING OF

IMPORTANT ACTIVITY” Submitted to the, in partial Fulfilment of the requirement for the
award of the degree of BACHELOR OF SCIENCE IN BIOCHEMISTRY is a record of original
research work done by me during 2023- 2024 under the supervision and guidance and it has
not formed the basis for the award of any Degree / Associate /Fellowship or similar Title to
any candidate of any university.

SIGNATURE OF THE CANDIDATE

 LIST OF CONTENTS
S.NO CONTENT PAGE
NUMBER

1 Abstract 01

2 Introduction 03

3 Aim and Objectives 14

4 Review of literature 16

5 Materials and Methods 20

7 Result and Discussion 33

8 Conclusion 37

9 Reference 39
ABSTRACT

1
ABSTRACT

Tabernaemontana divaricata (pinwheel flower) is a flowering plant that can grow

easily in gardens and along roadsides. This plant can be cultivated in every
condition. No specific environmental condition is required for the growth of the
plant. Growing evidence suggests that this plant has medicinal benefits for various
diseases due to the presence of bioactive components in the plant. The plant is
extensively found near Indian heritage to be used for worship. Their major
pharmacological potential is against inflammation, pain, and other diseases. Plants’
major activities, such as anti-diabetic, anti-inflammatory, antibacterial, antifungal,
and so on, have been demonstrated by their responsible bioactive compounds. The
review is to highlight the researchers’ findings of different medicinal activities in
T. Divaricata along with the major responsible phytocomponents. There is a lot

more scope for further research, which can be extended by the help of this review .
Qualitative phytochemical analysis was performed to identify the presence of
various phytoconstituents with standard procedures. Total phenols, total flavonoids
and total protein were determined by folin-ciocaltaeu method, aluminium chloride
colorimetric method and Lowry’s method. Phytochemical screening exhibits the
occurrence of alkaloids, flavonoids, terpenoids, cardiac glycosides, saponins,
tannins, carbohydrates and protein. In quantitative analysis, elevated level of
phenols, flavonoids and protein were identified in leaves, flowers and stem.

KEYWORDS: Alkaloids, Bioactive Components, Latex, Pharmacological

Activity, T. Divaricata.

2
INTRODUCTION

3
 INTRODUCTION

The plant which has medicinal value was always invaluable for humans and as the
time passes, its utility will also increase. Compound that are found naturally are
taken as safer in comparison to synthetic compound. Plants with medicinal value
are more difficult to develop drug resistance than synthetic compounds1. The plant
Tabernaemontana divaricata also known as crepe jasmine and pinwheel flower, is
from the family Apocynaceae. A plant that grows in spring is as graceful as it is
evergreen. This plant can be found throughout South Asia as well as the countries
of Southeast Asia. It is considered an ornamental plant and is abundantly found in
Indian heritage to worship the god and goddess. The phytochemical, nonalkaloids
and alkaloids constituents such as flavonoids, phenylpropanoids, terpenoids,
enzymes and steroids from the parts of the plant (stem, root, flower and leaves)
have been reported.

Along with its well-known analgesic and antidiarrheal properties, plant parts are
also used as liver, spleen, and brain tonics; and it has been discovered that T.
Divaricata extract has antioxidant, anti-inflammatory, and reversible
acetylcholinesterase inhibition properties2-4. A sticky milky liquid called latex
comes out from the points of laticiferous tissue and contains secondary metabolites
and proteins5-8. A milky fluid found in nature in 10% of all flowering plants is
Latex6. The plants of the family Apocynaceae have latex as one of their pertinent
features9. The easy and plentily availability of T. Divaricata as an ornamental plant
and its medicinal importance created this review. In the present review, an attempt
has been made to enlist the important scientifically proven pharmacological
applications along with the major phytoconstituents responsible for these activities
of T. Divaricata.

Nature is a warehouse for various plants with medicinal properties[1]. Many

varieties of medicinal plants are distributed worldwide but now only
4
80% of people trust conventional medicines made from plants for their
primary care of health [2]. It can be used to treat all diseases because of its
least or no side effects in the human being[3]. Medicinal importance of the
plants depends on the presence of some phytochemical constituents. These
phytochemical substances in the medicinal plants can generate physiological
consequences in the human body system [4, 5]. It can be derived from diverse
parts of the plants like flowers, leaves, stem and root to exploit against
disorders. There are two forms of phytochemicals. Primary metabolites are
amino acids, proteins, sugars and chlorophyll, secondary metabolites
comprises of phenols, alkaloids, flavonoids, terpenoids, tannins, saponins and
glycosides [6].
Tabernaemontana divaricata is a medicinal as fine as ornamental plant
belonging to the family of Apocynaceae shown in fig. 1. In various languages, it
is known as Crepe jasmine (English), Chandani (Hindi), Nandivardhanamu
(Telugu), Nandibatlu (Kannadam), Kutampale (Malayalam) and Nandiyavattai
(Tamil) [7, 8, 9]. Flowers are milky white in colour, cooling and fragrant. It can be
utilized to heal dermatopathy, flaming sense and ophthalmic problems [10]. The
leaves, flowers and stem of the plant are shown in the fig. 2, fig. 3 and fig. 4
respectively. They have been utilized habitually for the healing of ulcers and
rheumatism [11]. Root’s flavour is bitter and squashing of root reduces tooth ache.
It is used to avert inflammation when combine with water in sores [12].
T.divaricata possesses extensive series of useful actions like anti-infection, anti-
inflammation, anti-cancer, astringent, analgesic, antioxidant[13,14], anxiolytic, anti
diabetic and anticonvulsant [15- 17].

T. divaricata, also known as pinwheel flower, is commonly found in small trees

throughout South Asia10,11. One of the investigators dug out the workings of

5
the parts of the plant like stems and flowers to root extracts and extracts of leaves
of T. Divaricata. Moreover, the researcher screened 19,20- Dihydroervahanine and
19,20-Dihydrotabernamine along with isolated compounds like tabernaelegantine
A. It gave out the result that the respective compounds’ extracts show slightly
higher anti-acetylcholinesterase activity. Moreover, it has been shown in studies
that the compounds which were isolated from T. Divaricata show action against
cell lines and the plant also contains many non-alkaloid compounds like enzymes,
terpenoids and phenolic acid (Figure 1)12. 12-hydroxy akuammicine is a major
alkaloid of T. Divaricata. One of the investigators administered intravenously.
The IP administration in mice and rats of 12-hydroxy akuammicine stopped
the growth of ascites and alveolar lymphoma, at a concentration of 15-20
mg/kg/day for 10-20 days13. The alkaloids mentioned above are generally seen
in the roots of T. Divaricata. Acetylcholinesterase activity can be witnessed in
these alkaloids. The inhibitory effect of the alkaloids was proved to be specific and
competitive, along with being capable of getting back to its previous position14.
Tabernaemontana divaricata is a plant under the family Apocynaceae. It was
named by a German physician and botanist, J. Thomson Muller [1].
Approximately 100 species of this genus are currently found in Africa, Asia,
Oceania, and the Americas [2,3]. The plant can be found in India’s upper
Gangetic plain, Garhwal, East Bengal, Kasia Hills, Assam, Burma, and
Vishakhapatnam hills [4]. Plants which are commonly referred to as
“milkweed” due to their latex content.”. Other synonyms for it are Ervatamia
coronaria, Ervatamia microphyll, Ervatamia divaricata, Tabernaemontana
coronaria etc. The plant is also known as “Crape Jasmine” and “Chandani”
[5,6]. It is a 1.8-2.4 m tall evergreen shrub with silvery grey bark and milky
latex. The leaves are simple, opposite, elliptic or elliptic – lanceolate, smooth,
glossy green, acuminate and with curly margins (Figure 1). This plant
blossoms all year, having the most bloom in the spring and summer. It
features waxy, pure white, ruffled-edged blossoms that are,
6
 especially fragrant at night and after dark. They have 1-8 flowered cymes
at the branch bifurcations, with the corolla lobe overlapping to the right in
the bud. Fruit follicles are 2.5-7.5 cm long, ribbed and curved, orange or bright
red within narrowed into a slender curved beak. Seeds are dull brown, minutely
pitted, irregular, and enclosed in a red pulpy aril [7]. Plants in the genus
Tabernaemontana also have high alkaloid content and are often
pharmacologically active [8]. Furthermore, monoterpene indole and bis indole
alkaloids are the major classes of alkaloids within the genus.

Terpenes, lactones, steroids, phenolics, and flavonoids are among the other
compounds. Over 67 species have been investigated for indole alkaloids, of
which 470 isolations of approximately 240 structurally different bases have
been detected [2,9,10]. When used in small quantities, alkaloids are organic
products of natural or synthetic origin that are basic in nature and contain one
or more nitrogen atoms, usually of heterocyclic nature, and have specific
physiological actions on the human or animal body. There are many indole
alkaloids and their derivatives whose pharmacological activities are yet to be
studied [11]. Flowers of plant contain α – amyrin acetate, βamyrin acetate,
lupeol, β–sitosterol, stigmasterol and several other alkaloids like dregamine,
20-epiervatamine, tabernamontanine, vobasine, voacangine, voacamine,
flavonoid aglycones and flavanol glycosides like isovoacristine,
voaphyllinehydroxyindolenine, janetine (tetrahydrolivadine), N-
methylvoaphylline (hecubine), kaempferol [12,13]. Leaves of the plant contain
the same alkaloids present in the flowers as well as others like α –amyrin,
lupeol and their acetates, β-sitosterol, coronaridine, apparicine, ervaticine (2-
acyl indole derivative), ervaticine, hyderabadine, lahoricine, mehranine,
stapfinine, voacristine, voharine and a dimeric alkaloid, conophylline 17-
oestradiol [14]. Roots of plant contain 2 vobasinyliboga bisindole alkaloids i.e.,

7
 dihydroervahanin
e having anticholinesterase activity [15]. As antiparasitic against worms,
dysentery, diarrhoea, and malaria. Van Beek., et al. In western India
claim that the latex is used for inflammation and wound healing [16]. The ash
of the stem is used to treat ocular problems, the root to treat toothaches, and
milky latex infused with coconut oil to treat headaches. The blossoms are
soothing, aromatic, and they can be used to treat burning sensations,
ophthalmitis, and dermatopathy [7]. The plant also has antioxidant,
anticancer, and analgesic properties, which make it useful in the treatment of
fractures, rheumatic pain, fever, and so on. It is used in many regions around
the world as a tonic for the kidney, liver, and spleen, as well as to cure
disorders such as pruritus, asthma, hysteria, paralysis, piles, rabies, ulceration,
vomiting, and so on [17]. It has been reported that alcoholic extracts of
Tabernaemontana divaricata root and stem have a very high inhibitory
activity against AchE, implying that it may be practitioners in the field for
many neurodegenerative disorders, particularly myasthenia gravis and
Alzheimer’s disease [16]. It has also been reported that the plant extract can
treat skin diseases, aches, scabies and eye diseases and act as an
antihypertensive, diuretic, emmenagogue, hair growth promoter, aphrodisiac
[17]

8
 Kingdom: Plantae

Clade: Tracheophytes

Clade: Angiosperms

Clade: Eudicots

Clade: Asterids

Order: Gentianales

Family: Apocynaceae

Genus: Tabernaemontana

Species: T. Divaricata

Vernacular Names

English: Crepe Jasmine, Pinwheel Flower Sanskrit: Nandivarksha Hindi:

Chandni, Tagar, Tagari Assamese: Kathanda Bengali: Tagar, Kath Mallika
Kannada: Nandi battalu, Nanjubattalu, Nandyaavarta Mizo: Par-arsi, Kelte
bengbehBattalu Tamil: Nandiarvattai Gujarati: Sagar Marathi: Ananta, Tagar
Malayalam: Kutampale Bangladesh: Dudhful/ Kath-Mallika.

Botanical Features

Tabernaemontana divaricata (Figure 1) is an evergreen medicinal shrub and

moderately fast-growing. It can grow asagarden plant, and also found on
roadsides, lawns, and human settled areas. The plant is a glabrous, 1.5- 2.5m in
height with silvery grey bark, wrinkled and milky latexexudes when wounded,
and has snow white colored fragrant flowers. The leaves are shiny deep green
in color [6].

10
Tabernaemontana divaricata is a plant under the family Apocynaceae. It was named
by a German physician and botanist, J. Thomson Muller [1]. Approximately 100
species of this genus are currently found in Africa, Asia, Oceania, and the Americas
[2,3]. The plant can be found in India’s upper Gangetic plain, Garhwal, East
Bengal, Kasia Hills, Assam, Burma, and Vishakhapatnam hills [4]. Plants which
are commonly referred to as “milkweed” due to their latex content.”. Other
synonyms for it are Ervatamia coronaria, Ervatamia microphyll, Ervatamia
divaricata, Tabernaemontan tocoronaria etc. The plant is also known as “Crape
Jasmine” and “Chandani” [5,6]. It is a 1.8-2.4 m tall evergreen shrub with silvery
grey bark and milky latex.

The leaves are simple, opposite, elliptic or elliptic – lanceolate, smooth, glossy
green, acuminate and with curly margins (Figure 1). This plant blossoms all year,
having the most bloom in the spring and summer. It features waxy, pure white,
ruffled-edged blossoms that are especially fragrant at night and after dark. They
have 1-8 flowered cymes at the branch bifurcations, with the corolla lobe
overlapping to the right in the bud. Fruit follicles are 2.5-7.5 cm long, ribbed and
curved, orange or bright red within narrowed into a slender curved beak. Seeds are
dull brown, minutely pitted, irregular, and enclosed in a red pulpy aril [7].

11
 Plants in the genus Tabernaemontana also have high alkaloid content and often
pharmacologically active [8]. Furthermore, monoterpene indole and bis indole
alkaloids are the major classes of alkaloids within the genus. Terpenes, lactones,
steroids, phenolics, and flavonoids are among the other compounds. Over 67
species have been investigated for indole alkaloids, of which 470 isolations of
approximately 240 structurally different bases have been detected [2,9,10]. When
used in small quantities, alkaloids are organic products of natural or synthetic origin
that are basic in nature and contain one or more nitrogen atoms, usually of
heterocyclic nature, and have specific physiological actions on the human or animal
body.

There are many indole alkaloids and their derivatives whose pharmacological
activities are yet to be studied [11]. Flowers of plant contain α - amyrin acetate, β-
amyrin acetate, lupeol, β–sitosterol, stigmasterol and several other alkaloids like
dregamine, 20-epiervatamine, tabernamontanine, vobasine, voacangine, voacamine,
flavonoid aglycones and flavanol glycosides like isovoacristine,
voaphyllinehydroxyindolenine, janetine (tetrahydrolivadine), N-methylvoaphylline
(hecubine),kaempferol [12,13]. Leaves of the plant contain the same alkaloids
present in the flowers as well as others like α -amyrin, lupeol and their acetates, β-
sitosterol, coronaridine, apparicine, ervaticine (2-acyl indole derivative), ervaticine,
hyderabadine, lahoricine,mehranine, stapfinine, voacristine, voharine and a dimeric
alkaloid conophylline 17-oestradiol [14]. Roots of plant contain 2 vobasinyl- iboga
bisindole alkaloids i.e., 19,20-dihydrotabernamine and 19,20 dihydroervahanine
having anticholinesterase activity [15].

12
 Almost all parts of the Crape Jasmine plant can be utilised to cure a variety of
ailments. Its crude extract is antimicrobial against infectious disorders like syphilis,
leprosy, and gonorrhoea, as well as antiparasitic against worms, dysentery,
diarrhoea, and malaria. Van Beek., et al. in western India claim that the latex is
used for inflammation and wound healing [16]. The ash of the stem is used to treat
ocular problems, the root to treat toothaches, and milky latex infused with coconut
oil to treat headaches. The blossoms are soothing, aromatic, and they can be used to
treat burning sensations, ophthalmitis, and dermatopathy [7]. The plant also has
antioxidant, anticancer, and analgesic properties, which make it useful in the
treatment of fractures, rheumatic pain, fever, and so on. It is used in many regions
around the world as a tonic for the kidney, liver, and spleen, as well as to cure
disorders such as pruritus, asthma, hysteria, paralysis, piles, rabies, ulceration,
vomiting, and so on [17].

It has been reported that alcoholic extracts of Tabernaemontana divaricata root

and stem have a very high inhibitory activity against AChE, implying that it may be
practitioners in the field for man neurodegenerative disorders, particularly
myasthenia gravis and Alzheimer’s disease [16]. It has also been reported that the
plant extract can treat skin diseases, aches, scabies and eye diseases and act as an
antihypertensive, diuretic, emmenagogue, hair growth promoter, aphrodisiac [17].

13
AIM AND OBJECTIVE

14
 AIM AND OBJECTIVES

Aim:

To study the phytochemical profiles of Euphorbia Hirta with two different solvent
extractions, such as Aqueous and Methanol.

Objectives:

Complete extraction of Euphrobia Hirat using non-polar, middle polar and polar
solvents in order to see the quality of primary phytochemicals present, and the
profiles of secondary phytochemical components.

Carry out the phytochemical screening of plant extracts for compounds such as,

❖ Alkaloids

❖ Terpenoids

❖0 Phenol and Tannins

❖ Sugar

❖ Saponins

❖ Flavonoids

❖ Carbohydrates

❖ Steroids
❖ Proteins.

15

REVIEW OF LITERATURE
 16
REVIEW OF LITERATURE

The genus Tabernaemontana is comprised of about 110 species of plants, which

are found in tropical and some subtropical regions of South America, Sub Saharan
Africa, and Asia. Extracts of these plants have been utilized in traditional medicine
for a variety of purposes, ranging from cleaning wounds to treating syphilis.These
extracts have also been used for non-medicinal purposes, including as poison arrow
ingredients and as birdlime latex. Chemical screenings of Tabernaemontana plants
revealed their uses are a result of their rich alkaloid content. Phytochemical
investigations of the genus since the mid 20th century have led to isolation of
hundreds of biologically- and structurally interesting secondary metabolite
alkaloids, many of which contain or are derived from indoles. The
chippiine/dippinine/tronocarpine group of metabolites presently consists of 14
indole alkaloids (1–14) isolated from Tabernaemontana plants. These alkaloids all
possess pentacyclic ring-systems and are biogenetically related (vide infra). The
first member of this family was isolated in 1985 by Van Beek et al. from the root
bark of the African understory tree T. africana, whose extracts had previously been
shown to have strong antimicrobial activity.3a This pentacyclic alkaloid, chippiine
(1) (Fig. 1), was named after the species from which it was isolated, T. chippii.
Two structural analogs of alkaloid 1 have subsequently been isolated: (−)-10,11-
demethoxychippiine (2) (Fig. 1) from the South American tree T. markgrafiana by
Torssell et al. in 1994and (−)-16-O methyl-10,11-demethoxychippiine from the
roots of the Thai plant T. pandacaqui by Takayama et al. in 2019. Antioxidants are
molecules or compounds that interrupt the movement of free radicals such as
hydrogen peroxide to regulate the process of autoxidation or do the same by
directly squeezing their formation43,44. Constituents of therapeutic plants like
phenolic diterpenes, carotenoids, volatile oils, flavonoids, phenolic acids and
anthocyanidins prove to be and are used as antioxidants45.

17

These compounds choose free radicals as their target by consuming molecules of

oxygen, donating molecules of hydrogen, acting as reducing agents, or breaking up
antioxidant

chains44,46. The various parts of species T. divaricata are made of a compound or

extract of methanol, ethanol, aqueous, petroleum ether, hexane, octyl benzoate or
octyl benzoic acid, chloroform, and digalactosyl deconate. The models used for the
activity are fluorescence recovery after photobleaching, 2,2-
dipphenylpicrylhydrazyl assay - in vitro
In 1999, Kam and Sim isolated two novel chippiine-related indole alkaloids, (+)-
dippinines A6and C7from the leaf and stem-bark extracts of Malaysian flowering
plant T. corymbosa, which had previously been found to contain anticarcinogenic
vobasinyl-Iboga bisindoles.

18

The following year, this group isolated from the stem-bark of the same plant
another member of this family, (+)-tronocarpine (3) (Fig. 1),9 which possesses a
modified seven membered lactam C-ring and an unsaturated cyclohexene E-ring.
Kam et al. later isolated (−)-dippinines B (4) (Fig. 1) and D in 2001 from the stem
and leaf extracts of this plant, respectively. 10 In 2014, Kong and coworkers isolated
10 new indole alkaloids from the leaves of T. corymbosa. These metabolites
included (+)-tabercarpamines A–J, of which two dimeric bisindoles (A–B) and
four monomeric indole alkaloids (G–J) contain chippiine-type skeletons. 11 Since
(+)-tabercarpamines C–F are more closely related to tabersonine-type alkaloids,
these metabolites are not considered in this review.
 19

MATERIALS AND METHODS

 20

MATERIALS AND METHODS

Extract preparation

Flowers, Leaves and Stem were collected from the plant then washed, shade dried
and powdered. For the preparation of ethanol extract, 30g of each powdered
materials had been extracted with 200ml absolute ethanol using soxhlet apparatus
for 24 hours. The solvent was dispersed under vacuum and dried extracts used for
further analysis. Each extracts were mixed with suitable amount of respective
solvent at the time of usage. This hot percolation method was done in the
department of Biochemistry, KMG college of Arts and Science, Vellore
Qualitative Phytochemical screening

Several chemical tests to ascertain different phytochemicals are follows [18-22]

Alkaloids :

Alkaloids were identified through Mayer’s test. In the test, the extracted residues
were dissolved in 2N Hydrochloric acid. The mixture was filtered and the filtrate
treated with a few drops of Mayer’s reagent. Formation of cream precipitate
indicates the presence of alkaloids.

Flavonoids :

Using alkaline reagent test, flavonoids were detected. During this test, extracts
treated with few drops of NaOH solution. After addition of dilute hydrochloric

21
acid, formed intense yellow colour, which becomes colourless, indicates the
presence of flavonoids.

Steroids :

Libermann Burchard test was used for the identification of steroids. Within the
test, few drops of acetic anhydride was added to the extract, boiled and cooled.
After cooling, concentrated H2SO4 also added to the sides of the tube. At the
junction of two layers a brown ring formation followed by upper layer turns into
green reveals the presence of steroids.

Terpenoids :
Terpenoids were found by means of salkowski test. In this test, extracts treated
with little drops of Conc. H2SO4 leads to yellow coloured lower layer formation
shows the existence of terpenoids.

Glycosides :

During the examination of glycosides, extracts added with 5ml of dilute H2SO4
on water bath. Filtrates neutralize with 0.1ml of 5% NaOH. 0.1ml of Fehling’s
solution A and B added until it becomes alkaline and keep in water bath for
2minutes. Development of red precipitate illustrates the occurrence of
glycosides.

Cardiac Glycosides :

In this experiment, small amount of extracts dissolved in 1 ml of glacial acetic acid

containing 1 drop of ferric chloride solution and 1ml of Conc.

22

sulphuric acid. Appearance of brown ring between the interface reveals the
presence of cardioids.

Saponins :

Using foam test, saponins were detected. In the examination, little amount of
extracts shake with water. If foam produced persists for 10minutes shows the
presence of saponins.

Tannins :

Tannins were identified through ferric chloride test. Within this test, small
amount of extracts dissolve in water separately. When extracts treated with 5%
dilute ferric chloride leads to the appearance of blue colour if hydrolysable
tannins are present. Condensed tannins are identified by the existence of green
colour.

Phenols :

During the time for the analysis of phenols, small volume of extracts dissolved in
alcohol and alcoholic ferric chloride added to the above mixtures. Violet colour
formation leads to the presence of phenols.

Carbohydrates :

Carbohydrates were found via Molisch’s test. In this test, distilled water was
used to dissolve extracts and filtered. Few drops of alcoholic α-naphthol

23

solution and few drops of conc. H2So4 added to the extracts containing test tubes
resulted in purple to violet colour ring at the junction of two layers reveals the
occurrence of carbohydrates.

Protein :

Using Biuret test, protein was ascertained. In this examination, with 2ml of
extracts, 1ml of 40% NaOH and 2 drops of 1% Copper sulphate (Cuso4) were
added. Appearance of violet colour shows the existence of protein.

Quantitative analysis
Phenols, Flavonoids and Protein were measured by means of diverse methods.
They are as follows [23-25]

Total Phenols :

Total phenols was analysed using Folin-Ciocaltaeu method. In 200 μl of Samples,

1ml of folin- ciocalteu reagent and 0.8 ml of sodium carbonate (7.5%) was added.
The tubes stand for 30 min. Absorption measured at 765 nm. Gallic acid was used
as standard. The total phenolic content expressed as gallic acid equivalents (GAE)
in milligram per gram extract.

Total flavonoids :

Total flavonoids was estimated by aluminium chloride colorimetric method. In this

test, extracts (50μl) added to 4950 μl of distilled water and mixed with 0.3 ml of 5
% NaNO2.

24

This mixture incubated for 5 min at room temperature and 0.3 ml of 10 % AlCl3
was added. After 6. minutes of incubation, 2 ml of 1M

NaOH was combined followed by the addition of 2.4 ml distilled water.

Absorbance taken at 510 nm. Quercetin was used as standard.

Total Protein :

Total protein was calculated via Lowry’s method. In this procedure, with
different concentrations of standards, 0.1 ml of extracts was added in the
separate tubes and volume made up to 1ml with distilled water. In all the
tubes, 5 ml of alkaline copper sulphate reagent was mixed and left at room
temperature for 10 min. Then 0.5 ml of folin - ciocalteau reagent was
dissolved and incubated at room temperature for 20 min. The colour
developed read at 660 nm and the protein concentration expressed as mg per
gm extract.

Pharmacological activity

Antioxidant Activity : Antioxidants are molecules or compounds that interrupt the

movement of free radicals such as hydrogen peroxide to regulate the process of
autoxidation or do the same by directly squeezing their formation43,44. Constituents of
therapeutic plants like phenolic diterpenes, carotenoids, volatile oils, flavonoids, phenolic
acids and anthocyanidins prove to be and are used as antioxidants45.

25

These compounds choose free radicals as their target by consuming molecules of

oxygen, donating molecules of hydrogen, acting as reducing agents, or breaking up
antioxidant chains44,46. The various parts of species T. divaricata are made

of a compound or extract of methanol, ethanol, aqueous, petroleum ether, hexane,

octyl benzoate or octyl benzoic acid, chloroform, and digalactosyl deconate. The
models used for the activity are fluorescence recovery after photobleaching, 2,2-
dipphenylpicrylhydrazyl assay - in vitro, H2O2 free radicals, minimising power –
in vitroalong with superoxide anion radical scavenging, NO, Trolox equivalent
antioxidant capacity assay), H2O2 scavenging, and Aβ25 - 35 peptides, NOR
crystal violet assay and LPO assay47-58.

Anti-inflammatory Activity :

Inflammation is defined as the process by which an organism’s body responds to

an injury. Inflammation’s development is often a result of infection, chemical or
physical injury, injury to cells, and death59,60. One of the researchers investigated
the anti-inflammatory activity of flower extract obtained from T. divaricata.
Models were prone to chronic formalin and acute carrageenan61. The parts such as
leaves, stems, flowers, and other parts of the plant are made into compounds with
ethyl acetate, aqueous, ethanol, methanol and hexane fraction. The models this
activity uses are: minimization of interleukin-6 secretion and TNF production;
carrageenan and formalin prompt-mice models. The models of mice along with
croton oil bring on oedema in the models of mice53,55,61-63.

26

Anti-microbial Activity :

Compounds that are complex and do not allow the development of microorganisms
at petite assemblage are called antimicrobials64. Antimicrobials are often
described as secondary metabolites. They are more or less regularly created and
extracted either from therapeutic plants or even from microorganisms65. One of
the researchers has proved T. divaricata extracts work as antibiotics.
Monoterpenoid indole alkaloids, like voacamine type and 3- hydroxyiboga, are the
compounds that are activated biologically and perform the function of
antimicrobial agents, holding back the growth of bacteria, parasites and fungi66.

Antifungal Activity :

One of the researchers investigated and reported the antifungal activity of a

compound that is pharmacologically active from T. divaricata. Coronaridine, a
major compound from the plant T. divaricata, was found and cleared off from the
method - ethanolic extraction of plant T. divaricata67. The parts flower and leaves
of the plant make a compound or extract of ethanol and methanol along with
aqueous. The cell lines used for activity are Penicillium chrysogenum and
Malassezia furfur, and the method which is used for antifungal activity is the
Poisoned food technique in vitro68-70.

27

Anticancer Activity :

Various leaf extracts in the study were made in a solvent containing methanol and
ethyl acetate, along with chloroform and hexane, and were checked against these
cell lines: HT-29-Human colorectal adenocarcinoma cell line, 502,713, HCT-15 is
used for the colon, Michigan Cancer Foundation-7 (MCF-7) is for breast cancer ,
and Human prostatic carcinoma cell line (PC-3) is for prostate cancer40. Different
parts of the plant T. divaricata are made into an extract with different compounds
like ethanol, hexane, etc. for anti-cancer activity (Figure 2). Different parts of the
plant T. divaricata have several compounds that show anti-cancer activity. Root
bark of plant contains 5 and 3- oxocoronidine, ibogamine, leaves of plant contains
tabernaemontamine, mehranine, vocangine, voaphylline, and the root of plant
contains major 2 alkaloid which is conodurine and tabernaelegantine. For
anticancer activity, various cell lines were used, including HCT 15 (isolated from a
cancer patient’s large intestine), col-2 and HT-29 for colon cancer, S-10 for
sarcoma, V79 and LUP for lung cancer, Human Leukaemia-60, P-388, MOLT-4
(T-cell derived cell line) for blood cancer, and ZR-75-1 and BC- for breast cancer.
It has been scientifically proven that conophylline of T. divaricata inhibits the
stellate cells from α-SMA (α smooth muscle actin) and collagen production and
protects them from fibrosis67,71-84.

Acetylcholinesterase Activity :

One of the investigators, Ingkaninan et al., found the acetylcholinesterase activity

of T. divaricata’s methanolic extracts. Rats were used in the study as test models
and were tested in vivo85. Almost all parts of the plant show

28

acetylcholinesterase activity, but latex, flowers and roots show high

acetylcholinesterase activity. These parts of the plant make an extract with
different compounds like methyl alcohol, ethyl alcohol and PBS. There are several
compounds present in plants which are responsible for AChE activity, like 19,20
dihydro tabernamine, conodurinbe, nitrogen-methyvofinine, conophylline etc. The
method used for the activity is Ellman’s method71,86-91.

Acetylcholinesterase Activity :

One of the investigators, Ingkaninan et al., found the acetylcholinesterase activity

of T. divaricata’s methanolic extracts. Rats were used in the study as test models
and were tested in vivo85. Almost all parts of the plant show acetylcholinesterase
activity, but latex, flowers and roots show high acetylcholinesterase activity. These
parts of the plant make an extract with different compounds like methyl alcohol,
ethyl alcohol and PBS. There are several compounds present in plants which are
responsible for AChE activity, like 19,20 dihydro tabernamine, conodurinbe,
nitrogen-methyvofinine, conophylline etc. The method used for the activity is
Ellman’s method71,86-91

Anti-fertility Activity :

An anti-fertility activity is shown in the ethanolic extract of T. divaricata in

oestrogenic activity models in immature female rats. The rats that were used in this
study were immature female Albino Wistar. Researchers performed the experiment
and found the extract showed the existence of several compounds which have
medicinal value. The compounds are carbohydrates, steroids, alkaloids, glycosides,
flavonoids, tannins, and increased the uterus’ weight

29

tremendously. Some histopathological studies also showed normal architecture of

the uterus in vehicle-treated rats. It tells us about the surface epithelium that has
next to no secretory activity92.
Anticonvulsant Activity :

The plant is rich in alkaloids which have neurological activities like coronaridine,
dregamine, ibogamine and many other alkaloids. Khan and Mukhram did the
research for anti-convulsant activity of T. divaricata and found that the flowers of
the plant have some compounds that are responsible for anti-convulsant activity.
Researchers selected 2 models for the activity: the PTZ and MES induced
convulsion method and the compounds epivocangine, vobasine, voaphylline and
dihydro pericyclivine showed anti-seizure activity in rats and mice by using MES
induced convulsions and the PTZ induced convulsions method. Researchers found
that the methanolic extract of flowers can stop seizures in animals. It is due to the
inhibition of sodium channels, but the exact mechanism is not known93-101.

Anti-diabetic Activity :

The activity of T. divaricata (antidiabetic) from the extract (methanolic) was

applied to alloxan-prompted diabetic rats. The results that came out showed a great
amount of antidiabetic activity. Moreover, an additional decrease can also be seen
in the oxidative damage effect that can be seen in rats120. Kanthlal et al. suggest in
their study that the insulin receptors may be alerted by an extract (methanolic).
Henceforth, it may result in the production of beta-stem cells in the subject of the
test’s pancreas. Antidiabetic activity can be seen by the

30

compound conophylline, which is generally segregated from T. divaricata120,121.

Increased plasma levels in diabetic rats were observed in the study. A severe
decline in blood glucose levels can also be seen in the results, which indicates
antidiabetic activity as well122.

Anthelminthic Activity :

Helminths are commonly found infectious agents of humans in the developing

world. The disease caused by them creates such chaos across the world that it is
becoming difficult for countries to cope with it115. T. divaricata is one of the
Indian medicinal plants that shows anti-helminthic activity116. The anti helminthic
activity was performed on a mature earth worm found in India called Pheretima
posthuman. It was done so because it has a great resemblance not only
anatomically but also physiologically with the human being’s intestinal round
worm parasites117. Chloroform and methanolic extracts of T. divaricata leaves
were prepared, Radhika B and Vilasini S performed the experiment and observed
anthelmintic activity of methanol extract is more potent compared to the
chloroform extract115.

Gastrointestinal Effect :

Khan et al. found the gastro and intestinal effects of extract with methanol of T.
divaricata flowers. To evaluate the possible effects in the study, the model of a rat
pyloric ligation method (gastric ulceration induced) and as a standard drug one of
the proton-pump inhibitors was used. The study resulted in the discovery that the
extract reduced the gastric juice amount, free and total acidities, and ulcer index,
along with the pH of gastric acid produced118. Khan et al. also did one more test
using a range of concentrations of the methanol extract from the

31
T. divaricata flower. After measuring, parameters such as catalase and superoxide
dismutase, along with the mucin and total protein, when treated with extracts,
displayed an index that declined119.
 32

Result and Discussion

 33

RESULT

Phytochemical screening of Tabernaemontana divaricata

Phytochemical analysis of Tabernaemontana divaricata the presence of Alkaloids,
Flavonoids, Phenols, Tannis, Saponins, Carbohydrate, Protein in maximum
amount. Methonal extracts of leaves showed good results than aqueous extracts of
leaves in analyzing the bio active compounds.
This above image shows that phytochemical screening of Tabernaemontana
divaricata ( AqueousExtract )

This above image shows that phytochemical screening of Tabernaemontana

divaricata ( Methanol Extract )

34
Table: 1 (Phytochemical constituents Tabernaemontana divaricata of Leaves)
Phytochemical Aqueous Methanol
Test Extract Extract

Alkaloids + _

Terpenoids + +

Phenols and + _
Tannins
Sugar + +

Saponins + +

Flavonoids + +

Protein + +

Steroids _ _

Carbohydrates + _

35
Discussion

Phytochemical screening revealed the presence of alkaloids, flavonoids, steroids,

saponins, terpenes, and phenolic compounds in TD, so it may be possible that the
mechanism of anti-compulsive action of TD may be due to involvement of any of
these phytoconstituents in serotonergic neurotransmission. Moreover,
triterpepenoids (steroidal compounds), which are able to cross blood brain barrier
(BBB) due to their lipophilic nature, are present in the TD leaves. Hence, the
observed effect of TD in obsessive compulsive disorder could be related to
serotonergic modulation and anticompulsive activity at a molecular level in central
nervous system. In conclusion, ethanolic extract of TD leaves and FLX, reduced
obsessive and compulsive symptoms and produced antianxiety effects.

The presence of several phytochemical components and pharmacological

properties of Tabernaemontana divaricata are discussed in this review. The
pharmacological activities reported in this review confirm that this plant have a
high therapeutic value, with a leading capacity for the future development of a
new, safe and effective herbal remedy. The pharmacological activities of this plant
may be due to alkaloids and non-alkaloids isolated from it. Reported research has
found that bioactive monoterpene indole alkaloids derived from nature play a vital
role in human health, nutrition, and disease prevention. Furthermore, bioactive
components have demonstrated a wide range of biological effects, including
antimicrobial, antioxidant, anti-inflammatory, anticholinesterase, anticancer, anti-
diabetic, anti-hypertensive, anti-fungal, wound healing, and analgesic effects.

36
 CONCLUSION

37

CONCLUSION

In the present study, phytochemical analysis of chemical constituents and

standardization of plant extracts of Tabernaemontana divaricata was done,
Tabernaemontana divaricata is the wellknown medicinal plant used from the
ancient era to till date for their medicinal values. Standardization was done on the
basis of marker compounds isolated from the plant extracts. The plant was
collected and analyzed.

38
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39
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