ABSTRACT

The present study deals with the pharmacognostic and phytochemical studies of leaves of
Ocimum americanum. It includes collection, identification, microscopical and phytochemical
evaluation of leaves of O. americanum. The preliminary phytochemical studies indicate the
presence of volatile oils, flavonoids, carbohydrates, phytosterols, tannins and fixed oils. The
sections were taken and cellular structures were studied. The T.S of leaf shows the presence
of epidermis, parenchymatous cells, collateral vascular bundle, lateral vein, glandular
trichomes which are peltate type and sessile. Powder microscopy of the leaf shows the
presence of epidermal peelings and multicellular unbranched trichomes.
 INTRODUCTION
Ocimum americanum Linn commonly called as Ocimum Canum belongs to the Family:
Lamiaceae (Labiatae) .it is generally distributed throughout India, in fields of waste lands,
Plains and lower hills of India. It is Common in wastelands, by arable lands; and
paleotropic.The plant is a pubescent erect much branched herb, 15-60 cm high with
subquadrangular striate branches (1). Leaves are elliptic-lanceolate, entire, glabrous and
gland dotted strongly aromatic herb; branchlets puberulous, terete to four- angular. Leaves
are ellipticlanceolate, entire or faintly toothed, almost glabrous, gland-dotted. Flowers are
small, white, pink or purplish, in more or less closely set whorls in spiciform racemes.Seeds
are having Nutlets with narrowly ellipsoid, punctulate black (2). The main chemical
constituents are Volatile oils include methyl cinnamate, methylheptenone,
methylnonylketone, d-camphor, citral, Ocimin, methylchavicol, linalool, nevadensin,
salvigenin, beta-sitosterol, betulinic, ursolic, oleanolic acids, flavanoids, pectolinarigenin-7-
methylether and nevadensin. Polysaccharides (1). The Ocimum (Basil). comprises some of
the most popular herbs in the world. It belongs to the family Lamiaceae, subfamily
ocimoideae and includes more than150 different species and varieties distributed in the
tropical regions of Asia, Africa, and South Africa considered as one of the largest genera of
the Lamiaceae family. The name Tulsi is a Sanskrit word which means “matchless one.”6,7
Among the plants known for medicinal value, the plants of genus Ocimum are very important
for their therapeutic potentials. Because of its popularity basil is often referred to as King of
herbs, being widely utilized due to its economic, nutritional, industrial and medicinal
properties.8-10 In traditional systems of medicine, different parts (leaves, stem, flower, root,
seeds and even whole plant) of O. sanctum have been recommended for the treatment of
bronchitis, bronchial asthma malaria, diarrhoea, dysentery, skin diseases, arthritis, chronic
fever and antifungals. and anti-diabetic Tulsi consists of dried whole plant of Ocimum
sanctum Linn (3). (Fam. Lamiaceae); an erect, annual herb 30 - 60 cm height, much
branched, found throughout the country. In the present investigation Ocimum
tenuiflorum (O.santum) have been selected for the standardization studies due to
their medicinal importance (4).
2:- Review of literature
Standardization: standardization of drug means conformation of its identity and
determination of its. quality and purity and detention of nature of adulterant by various
parameters like morphological. quality and purity and detention of nature of adulterant by
various parameters like morphological. quality and purity and detention of nature of
adulterant by various parameters like. microscopical, physical, chemical and biological
observation. The plant environment and genetic. factors could significantly affect the
biochemical components of the plant extract, in which plants are still the most abundant and
cost-effective resource for drug innovation.

2.1. MORPHOLOGY OF TULSI-.

Ocimum sanctum L. (Tulsi) is an erect, much branched sub-shrub 30-60 cm tall, with simple
opposite green or purple leaves [Figure 1] that are strongly scented and hairy stems. Leaves
have petiole and are ovate, up to 5 cm long, usually somewhat toothed. Flowers are purplish
in elongate racemes in close whorls [Figure 2]. Tulsi is native throughout the world tropics
and widespread as a cultivated plant and an escaped weed. It is cultivated for religious and
medicinal purposes and for its essential oil. Tulsi is an important symbol in many Hindu
religious traditions, which link the plant with Goddess figure. The name ‘Tulsi in Sanskrit
means ‘the incomparable one’. The presence of a Tulsi plant symbolizes the religious bend of
a Hindu family (5).

Fig 1 : Ocimum sanctum (Tulsi) Fig 2 : Leaves of Ocimum sanctum

2.2. Macroscopic characters-;

Root - Thin, wiry, branched, hairy, soft, blackish-brown externally and pale, violet internally.
Stem - Erect, herbaceous, woody, branched; hairy, sub quadrangular external purplish brow
to black, internally cream, coloured; fracture, fibrous in bark and short in xylem; odour
faintly aromatic (6).

Leaf - 2.5-5 cm long 1.6 - 3.2 cm wide, elliptic oblong, obtuse or acute, entire or serrate,
pubescent on both sides; petiole thin, about 1.5-3 cm long hairy; odour, aromatic; taste,
characteristic
.
Flower - Purplish or crimson coloured, small in close whorls; bracts about 3 mm long and
broad, pedicels longer than calyx, slender, pubescent; calyx ovoid or campanulate 3-4 mm
bilipped, upper lip broadly obovate or suborbicular, shortly apiculate, lower lip longer than
upper having four mucronate teeth, lateral two short and central two largest; corolla about 4
mm long, pubescent; odour, aromatic; taste, pungent (7).

Fruit - A group of 4 nutlets, each with one seed, enclosed in an enlarged, membranous,
veined calyx, nutlets sub-globose or broadly elliptic, slightly compressed, nearly smooth; pale
brown or reddish with small black marking at the place of attachment to the thalamus; odour,
aromatic; taste, pungent (8).

Seed - Rounded to oval; brown, mucilaginous when soaked in water, 0.1 cm long, slightly
notched at the base; no odour; taste, pungent, slightly mucilaginous.

b) Microscopic of Stem
Shows a single layered epidermis with uniseriate, multicellular covering trichomer having 5-6
cells, occasionally a few cells collapsed; cortex consists of 10 or more layers of thin-walled,
rectangula parenchymatous cells; phloem consists of sieve elements, thin-walled, rectangular
parenchyma cells and fibres; fibres found scattered mostly throughout phloem, in groups and
rarely in singles; xylem occupies major portion of stem consisting of vessels, tracheids fibres
and parenchyma; vessels pitted; fibres with pointed ends; centre occupied by narrow pith
consisting of round to oval, thin-walled, parenchymatous cells (9).
 Fig
er :- 3 - Diagrammatic Figer :- 4- Outer region

Figer 5 :-Inner region Figer 6 :- Middle region

C) Microscopic of Leaf
Petiole - shows somewhat cordate outline, consisting of single layered epidermis composed
of thin-walled, oval cells having a number of covering and glandular trichomes; covering
trichomes multicellular 1-8 celled long rarely slightly reflexed at tip; glandular trichomes
short, sessile with 1-2 celled stalk and 2-8 celled balloon-shaped head, measuring 22-27 in
dia epidermis followed by 1 or 2 layers and 2 or 3 layers of thin-walled, elongated,
parenchyma cells towards upper and lower surfaces respectively; three vascular bundles
situated centrally, middle one larger than other two; xylem surrounded by phloem (10).

Midrib - epidermis, trichomes and vascular bundles similar to those of petiole except cortical
layers reduced towards apical region
Lamina - epidermis and trichomes similar to those of petiole; palisade single layered
followed by 4-6 layers of closely packed spongy parenchyma with chloroplast and oleo-resin;
stomatal index 10-12-15 on upper surface and 14 - 15 - 16 on lower surface; palisade ratio
3.8; vein islet number 31 - 35.

Figer 7:- Vascular region Figer 8:- Mid-rib

2.3. PHARMACOLOGICAL ACTIVITY

Anticancer activity In ayurveda, various plants are used as a potential source of anticancer
and antitumor properties. It has been found that ethanolic extract of Ocimum sanctum
mediated a significant reduction in tumor cell size and an increase in lifespan of mice having
Sarcoma-180 solid tumors Ocimum has the ability to protect the DNA of the body from
dangerous radiations (11)

Antidiabetic activity
O. sanctum has been reported to possess very good anti diabetic Properties of the leaf powder
reduced fasting blood sugar, uronic acid, total amino acids, total cholesterol (TC),
triglyceride, phospholipids, and total lipids in normal and diabetic rats. TC and
triglycerides were dramatically reduced in the liver. Total lipids were dramatically
decreased in the liver and kidney (12)
Antilipidemic activity
Hyperlipidaemia, atherosclerosis and related diseases are becoming a major health problem
now days. Aqueous extract of O. sanctum reduces the level of total cholesterol, triglycerides
and LDL cholesterol levels in acute hyperlipidaemia induced by triton WR- 1339 in rats In a
study conducted on rabbits a diet supplemented with 1-2 % fresh leaves of Tulsi for 28 days
lowered the total lipid (9).
Antibacterial activity
Antibacterial activity of the aqueous, alcoholic, chloroform extract and oil obtained from
leaves of Ocimum sanctum were studied against E.coli, P. aeruginosa, S. typhimurium and S.
aureus. Extract obtained from O. sanctum were observed equally effective against pathogenic
gram-positive and gram- negative bacteria
Eye Disease
The leaf juice of Ocimum sanctum along with triphala is used in ayurvedic eye drop
preparations recommended for glaucoma, chronic conjunctivitis and other painful eye
disease. In daily routine one may use about three drops of tulsi oil along with honey and it is
supposed to improve eye sight (12)

Anti fertility activity

The benzene and petroleum ether extracts of leaves of Tulsi have been reported to produce
80% and 60% antifertility activity respectively in female rats In Kerala the local women as
well as the Ayurvedic physicians have been reported to use the leaves of Tulsi for antifertility
effect One of the major constituents of the Tulsi leaves is ursolic acid and it has been
reported that it possess anti fertility effect. This effect has been attributed to its anti-
estrogenic activity which mayberesponsible for arrest of spermatogenesis in males and due to
inhibitory effect on implantation of ovum in females.This constituent may prove to be a
promising anti-fertility agent devoid of side effects. In males, Tulsi leaves reduce
spermatogenesis by retarding sertoli cells activity (13)
3:-Aim and objectives

Aim: to perform standardization of Ocimum sanctum

Objectives: standardization helps in Identification and isolation of bioactive compounds

from the extract of Ocimum sanctum using various chemical and physical standardization.

4:- Materials and methods

4.1:-Collection of plant material: the tree of Ocimum sanctum was found in our
campus and the leaves were collected from that tree.

4.2:-Preparation of powder sample: The leaves were washed under running tap
water to remove the surface pollutants and then cut into little pieces, and air dried for several
days at room temperature. After being completely dried they were pulverized by grinder
machine to get powder and stored in an air tight container to prevent it from moisture and
avoid contamination. This powdered materials is used for further physiochemical and
phytochemical analysis (11).

4.3:- Physicochemical parameter:

4.3.1Extractive value: Amount of the active constituents present in crude drug.
Material when extracted with specific solvent. It is of two types: Ethanol soluble extract and
water soluble extract
4.3.1.1 Ethanol soluble extractive: (IP 2010) Macerate 5 g of the air-dried
drug, coarsely powdered, with 100 m1 of ethanol of specified strength in a closed flask for 24
hours shaking frequently during the first 6 hours and allowing to stand for 18 hours.
Thereafter filter rapidly taking Precautions against loss of ethanol evaporate 25 m1 of the
filtrate to dryness in a tarred flat-bottomed shallow dish, dry at 14105° and weigh. Calculate
the percentage of ethanol soluble extractive with reference to the air-dried drug (7).

4.3.1.2 Water soluble extractive:(IP 2010) Macerate 5 g of the air-dried

drug, coarsely powdered, with 100 m1 of chloroform water of specified strength in a closed
flask for 24 hours, shaking frequently during the first 6 hours and allowing to stand for 18
hours. Thereafter, filter rapidly taking Precautions against loss of water, evaporate 25 m1 of
the filtrate to dryness in a tarred flat-bottomed shallow dish, dry at 105° and weigh. Calculate
the percentage of water-soluble extractive with reference to the air-dried drug.
4.3.2 Loss on drying :( IP 2010) Loss on drying is the loss of weight expressed as
percentage w/w resulting from water and volatile matter of any kind that can be driven off.
Under specified conditions. The test is carried out on a well-mixed sample of the substance.
If the substance is in the form of large crystals, reduce the size by rapid crushing to a powder.
Where the drying temperature is indicated by a single value other than a range, drying is
carried out at the prescribed temperature ± 2°. Unless otherwise specified in the individual
monograph Weigh a glass-stoppered, shallow weighing bottle that has been dried under the
same conditions to be employed in the determination. Transfer to the bottle the quantity of
the sample specified in individual monograph, cover it and accurately weigh the bottle and
the contents. Distribute the sample as. evenly as practicable by gentle sidewise shaking to a
depth not exceeding 10 mm. Dry the substance by placing the loaded bottle in the drying
chamber as directed in monograph, remove the stopper and leave it also in the chamber. Dry
the sample to constant weight or for the specified time and at the temperature indicated in the
monograph. Dry by one of the following procedures. After drying is completed, open the
drying chamber, close the bottle promptly and allow it to cool to room temperature (where
applicable) in a desiccator before weighing. Weigh the bottle and the contents (10).
a) "In a desiccator": dry over phosphorus pent oxide at atmospheric pressure and at room
temperature.
b) "In an oven within a specified temperature range": dry in an oven within the range given in
the monograph;
Identification of the plant constituents by phytochemical tests:
Ethanolic extract, chloroform extract, pet ether extract and aqueous
extract are subjected to
various preliminary phytochemical analysis to test for the presence or
absence of various phytoconstituents by the following tests.
1. Test for alkaloids:
To the extract dilute hydrochloric acid will be added and filtered. The
filtrate will be treated with various alkaloidal reagents
a) Mayer’s test:
The filtrate will be treated with Mayer’s reagent: appearance of cream
colour indicates the presence of alkaloids.
b) Dragendroff’s test:
The filtrare will be treated with Dragendroffs reagent: appearance of
reddish brown precipitate
indicates the presence of alkaloids.
c) Hager’s test:
The filtrate when treated with Hager’s reagent, appearance of yellow
colour precipitate indicates the presence of alkaloids.
2) Test for carbohydrates and reducing sugar
The small quantities of the filtrate will be dissolved in 4ml of distilled
water and filtered. The
filtrate will be subjected to
a) Molisch’s test:
A small portion of the filtrate will be treated with Molisch’s reagent and
sulphuric acid. Formation of a violet ring indicates the presence of
carbohydrates.
b) Fehling’s test:
The extract will be treated with Fehling’s reagent A and B. The
appearance of reddish brown
colour precipitate indicates the presence of reducing sugar.
c) Benedict’s test:
The extract will be treated with Benedict’s reagent; appearance of reddish
orange colour precipitate indicates the presence of reducing sugar.
d) Barfoed’s test:
The extract will be treated with barfoed’s reagent and heated.
Appearance of reddish orange colour precipitate indicates the presence of
non reducing sugars.
3) Test for steroids:
Libermann burchard’s test:
The extract will be treated with 3ml of acetic anhydride, few drops of
glacial acetic acid followed by a drop of concentrated sulphuric acid.
Appearance of bluish green colour indicates
the presence of steroids.
4) Test for proteins:
a) Biuret test:
The extract will be treated with copper sulphate solution, followed by
addition of sodium hydroxide solution; appearance of violet colour
indicates the presence of proteins.
b) Millon’s test:
The extract will be treated with Millon’s reagent; appearance of pink
colour indicates the presence of proteins.
5) Test for tannins:
The extract will be treated with 10% lead acetate solution; appearance of
white precipitate indicates the presence of tannins.
6) Test for phenolic compounds:
a) The extract will be treated with neutral ferric chloride solution;
appearance of violet colour
indicates the presence of phenolic compounds.
b) The extract will be treated with 10% sodium chloride solution;
appearance of cream colour
indicates the presence of phenolic compounds.
7) Test for flavonoids:
a) 5ml of extract will be hydrolyzed with 10%sulphuric acid and cooled.
Then, it will be extracting with diethyl ether and divided in to three
portions in three separate test tubes. 1ml of diluted sodium carbonate,
1ml of 0.1N sodium hydroxide, and 1ml of strong ammonia solution will be
added to the first, second and third test tubes respectively. In each test
tube. Development of yellow colour demonstrated the presence of
flavonoids.
b) Shinoda’s test:
The extract will be dissolved in alcohol, to which few magnesium turnings
will beaded followed by concentrated HCL drop wise and heated, and
appearance of magenta colour shows the presence of flavonoids.
8. Test for gums and mucilage:
The extract was treated with 25 ml of absolute alcohol, and filtered. The
filtrate will examine for its swelling properties.
9. Test for glycosides
When a pinch the extract was treated with glacial acetic acid and few
drops of ferric chloride
solution, followed by the addition of conc. Sulphuric acid, formation of ring
at the junction of
two liquids indicates the presence of glycosides.
10. Test for saponins
Foam test
About 1 ml of the extract was diluted to 20 ml of with distilled water and
shaken well in a test
tube. The formation of foam in the upper part of test tube indicates the
presence of saponins.
11. Test for Triterpenoids
The substance was warmed with tin and thionyl chloride. Pink colour
indicates the presence of
triterpenoids.
5.1. Result:-
 Table 2. Loss on drying of Ocimum sanctum
Sr. No Loss on drying Result (%w/w)

1. Loss on drying 13.37%

5.2. Phytochemical screening:-

S. No Test Alcoholic Extract Aqueous Extract

1 alkaloids Absent Absent

2 carbohydrates Present Present

3 glycosides Absent Absent

4 phytosterols Absent Absent

5 fixed oils Absent Absent

6 saponins Absent Absent

7 tannins Present Absent

8 proteins and amino acids Absent Absent

9 gums and mucilage Absent Absent

10 flavanoids Present Present

11 terpenoids Absent Absent

Reference

1. Sarma DSK, Babu AVS. Pharmacognostic and phytochemical studies of Ocimum

americanum. J Chem Pharm Res. 2011;3(3):337–47.

2. Sarma DSK, Babu AVS. Antioxidant and Antimicrobial Activity of Ocimum

americanum. Int J Adv Pharm Sci. 2011;2:211–8.
3. Prabhavathi O, Yuvarajan R, Natarajan D. Mosquitocidal properties of Ocimum
canum Sims (Lamiaceae) leaf extracts against dengue vector Aedes aegypti L.(Diptera:
Culicidae). J Entomol Acarol Res. 2016;48(3):345–54.

4. Tewari BB, Gomathinayagam S. A critical review on Ocimum tenuflorum, Carica

papaya and Syzygium cumini: the medicinal flora of Guyana. Rev Boliv Química.
2014;31:28–41.

5. Mandal A, Reddy PJM. A review on phytochemical and pharmacological uses of

Ocimum sanctum linn. World J Pharm Res. 2015;4(7):1535–49.

6. Pattanayak P, Behera P, Das D, Panda SK. Ocimum sanctum Linn. A reservoir plant
for therapeutic applications: An overview. Pharmacogn Rev. 2010;4(7):95.

7. Rajalakshmi P, Vadivel V, Brindha P. Review on medicinal plants recommended in

Siddha literatures for the management of hypertension. Int J Res Pharm Sci.
2016;7(1):16–33.

8. Jeba CR, Vaidyanathan R, Rameshkumar G. Immunomodulatory activity of aqueous

extract of Ocimum sanctum in rat. Int J Pharm Biomed Res. 2011;2(1):33–8.

9. Wiart C. Anti-inflammatory plants. Ethnopharmacol Med plants Asia Pacific. 2007;1–

55.

10. Daly DC, Harley MM, Martínez-Habibe MC, Weeks A. Burseraceae. In: Flowering
Plants Eudicots: Sapindales, Cucurbitales, Myrtaceae. Springer; 2010. p. 76–104.

11. Singh N, Verma P, Pandey BR, Bhalla M. Therapeutic potential of Ocimum sanctum
in prevention and treatment of cancer and exposure to radiation: An overview. Int J
Pharm Sci drug Res. 2012;4(2):97–104.

12. Joseph B, Nair VM. Ocimum sanctum Linn.(Holy basil): pharmacology behind its
anti-cancerous effect. Int J Pharm Bio Sci. 2013;4(2):556–75.

13. Murkar S, Jadhav N, Veer S, Badhe P. Ocimum Sanctum Linn.(Tulsi): An

Ethnomedicinal Herb Used in the Prevention and Treatment of Cancer. Int J Pharm
Sci. 2023;8(6):1–12.