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International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
Volume III, Issue 3 March 2017
All rights are reserved 54
PHARMACOGNOSTICAL AND PHARMACOLOGICAL PROFILE OF
CRATAEGUS SONGRICA
* Rohit Mohan and * * Dr. Anil Middha
* Research Scholar, OPJS University, INDIA
* * Department of Pharmacy OPJS University, INDIA
Abstract
The Current research work showed the importance of herbal medicines in now a days, and it
also provide knowledge of standard parameters for quality control of herbal drugs. Some
parameters are given in the research are macroscopic and microscopic evaluation physic-
chemical parameters such as ash value, extractive values with different solvents, and for
evaluating efficacy of crude extracts biological acivity has been performed.
Introduction
GENERAL INTRODUCTION
History Traditional Medicines
Traditional medicines believe on the basis of knowledge and clinical experience of the
practitioners for indigenous systems of medicine. According to our history, many infectious
diseases have been treated with the help of herbals. The traditional medicine is increasingly
solicited through the traditional practitioners and herbalists in the treatment of infectious
diseases. Among the remedies used, plant drugs constitute an important part. Current status
on scientific investigations have highlighted the importance and the contribution of many
plant families i.e. Asteraceae, Liliaceae, Apocynaceae, Solanaceae, Caesalpinaceae,
Rutaceae, Piperaceae, Sapotaceae, Campanulacae, Erytrhoxylacae, Nyssaceae, Acanthacae,
Rubiaceae, Berberidaceaae, Graminae, Moraceae, Umbelliferae, Zingiberaceae,
Leguminasae, Pinaceae, Cucurbitaceae, Styraceae, Convolvulaceae, polypodiaceae. (Kokate,
C. K, 2006)
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
Volume III, Issue 3 March 2017
All rights are reserved 55
Ayurveda is the ancient Indian system of medicine for better life and longevity though
physical and mental health management. It involves a holistic approach of humans, their
health, and illness. The origin of Ayurveda has been lost in prehistoric antiquity, but its
characteristic concepts appear to have matured between 2500 B.C and 500 B.C. in India. The
word "Ayurveda" is derived from "Ayus (r)," meaning life, and "Veda," meaning knowledge;
thus, Ayurveda literally means "Science of life." It is the ancient Indian system of health care
and longevity. Ayurveda takes a holistic view of man, his health, and illness. It aims at
positive health, which has been defined as a well balanced metabolism coupled with a healthy
state of being. Disease, according to Ayurveda, can arise from the body and/or the mind
because of external factors or intrinsic causes. Ayurvedic treatment is aimed at the patient as
an organic whole, and treatment consists of salubrious use of drugs, diets, and certain
practices. Ayurveda has a vast literature in Sanskrit and various Indian languages, covering
various aspects of diseases, therapeutics, and Pharmacy. It has evolved its own theoretical
base, which is difficult to comprehend in terms of modern scientific concepts, at least at
present. However, here we are concerned only with the exploration of its materia medica
(Dev, S., 1988).
Pharmaceutics occupies an important place in Ayurveda. Medicinal preparations are complex
mixtures including plant and animal derived products, minerals, and metals. Plants form a
dominant part of Ayurvedic Pharmacopoeia. The earliest references to such plants are found
in the Rig Veda and the Atharva Veda, dating back to the second millennium B.C. The
Charaka Samhita (900 B.C.) is the first recorded treatise fully devoted to the concepts and
practice of Ayurveda; its primary focus was therapeutics. This work listed 341 plants and
plant products for use in medicine (Sharma, S. P, 1981). The next landmark in Ayurvedic
literature was the Sushruta Samhita (600 B.C.) (Krishnamurthy, K. H., 1999) which has
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
Volume III, Issue 3 March 2017
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special emphasis on surgery. It described 395 medicinal plants, 57 drugs of animal origin,
and 64 minerals and metals as therapeutic agents. Sushruta, the father of surgery, lived and
practiced surgery in Varanasi, India, approximately 2,500 years ago. Another important
authority in Ayurveda was Vagbhatta of Sind, in present day Pakistan, who practiced around
the seventh century A.D. His work Ashtanga Hridaya is considered unrivaled for the
principles and practice of medicine (Garde, G.K., 1954 )The Madhava Nidana (800-900 A.D)
was the next important milestone; it is the most famous Ayurvedic work on the diagnosis of
diseases. The last celebrated writer on Hindu medicine was Bhava Mishra of Magadha,
whose treatise Bhava Prakashan, is held in high esteem by modern Ayurvedic practitioners
for its descriptions of approximately 470 medicinal plants. Other than these monumental
treatises, many (>70) Nighantu Granthas (Pharmacy lexicons) were written, mostly between
the seventh and sixteenth centuries. Raj Nighantu by Narhari Pandita and Madanpala
Nighantu by Madanpala are considered masterpieces on medicinal plants.
All ancient texts on Ayurveda divide medical knowledge into eight branches (Ashtanga), and
this is no different from the contemporary approach. Also, Ayurvedic descriptions of diseases
are much like the modern delineation. Plant-derived drugs have been categorized according
to their pharmacologic/therapeutic action. For example, in the Charaka Samhita, drugs were
classified into 50 groups. Ayurveda, in its prime, was a congent, scientifically organized
discipline. Ayurvedic texts were much respected in neighboring countries, as evidenced from
their translation into Greek (300 B.C.), Tibetan and Chinese (300 A.D.), Persian and Arabic
(700 A.D.), and several languages of other Asian people. Currently, Ayurveda is widely
practiced in the Hindustan peninsula (India and the neighboring countries) and, in recent
years, has attracted much attention in economically developed countries such as those in
Europe and in the United States and Japan. ( Hartzell J. F; Zysk, K. G., 1995).
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
Volume III, Issue 3 March 2017
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Ayurvedic treatment of a disease consists of salubrious use of drugs, diets, and certain
practices. Medicinal preparations are invariably complex mixtures, based mostly on plant
products. Around 1,250 plants are currently used in various Ayurvedic formulations. Many
Indian medicinal plants have come under scientific scrutiny since the middle of the
nineteenth century, although in a sporadic fashion. The first significant contribution from
Ayurvedic materia medica came with the isolation of the hypertensive alkaloid from the
Sarpgandha plant (Rauwolfia serpentina), valued in Ayurveda for the treatment of
hypertension, insomnia, and insanity. This was the first important ancient-modem
concordance in Ayurvedic plants. With the gradual coming of age of chemistry and biology,
disciplines central to the study of biological activities of natural products, many Ayurvedic
plants have been reinvestigated. (Kokate, C. K, 2006).
PLANT INTRODUCTION
Plant Profile
Plant Profile of Crataegus Songorica
Table: 1 Nomenclature and taxonomical classification of Crataegus Songorica
Synonym Crataegus oxycantha
Family: Rosaceae
Genus: Crataegus
Species: Songorica
English name: Hawthorn
Local Ban-sangli, Ring [Kashmiri],Ban-Sangli [Punjabi]
Trade name: English Hawthorn Bansangli
Habit: Tree
Status: Common
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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Description of Natural Habitat
The plant grows in river valleys and rivine slopes in the-mountain zone in India, found in the
temperate Himmalayas of Kashmir and Himachal Pradesh, altitude of 1800-3000m.
[Nadkarni, 1976].
Distribution of plant
In Pakistan, the plant is found in mountains of Chitral [Boni], Swat, Chitral, Balochistan from
1300 to 2700 meters, This plant is exceptionally common in Boni [Chitral], Swat, Astor
Gilgit and Muree hilly areas with local names shetal or gain respectively; also distributed
from Afghnistan to Uttarr Pardesh 1500-2700m
Crataegus Songarica has about 200 spicesIt is cultivated in Asia, Australia, Canada. It is an
ornamental small hardy, spiny, deciduous tree or shrub up to 9 m tall, bark peeling off in long
flakes [Anwar. et al. 1979], armed with straight axillary's or terminal thorns; leaves deeply
and sharply irregularly 4¬7 lobed; the small rose- like white flowers are beautiful and
abundant [Wealth of India, 2003], in many flowered terminal corymbose cymes, emitting
spicy odour [Anwar. et al. 1979], the flowers have a foetid smell somewhat like decaying
fish; fruit red, globose, ovoid or scarlet or some times yellow, blue or black in colour,
containing 2-3 more or less united, bony Pyrenees[Nadkarnis, 1976].it is 16mmin diameter,
the flesh is mealy and dry, like that of rosehips [Anwar. et al. 1979; Shinwari .et al. 2006], the
fruit ripen in early to mid autumn [Brown, 1995].
The plant prefers light, medium and heavy soils and can grow in heavy clay soil .The plant
prefers acid, neutral and basic [alkaline] soil. It requires moist or wet soil and can tolerate
drought. The plant can tolerate strong winds but not maritime exposure. It can tolerate
atmospheric pollution
Medicinal Uses
The liquid extract of the flowers and fruits are used as heart tonic, improves blood flow, in
the coronary arteries and used in functional diseases of the heart, such as dysproea,
hypertrophy, vulvular insufficiency and heart oppression, arteriosclerosis and angina pectoris
[Shinwari, 2006; Nadkarnis, 1976; Kiritikar, 1918]. The plant is reported to be astringent,
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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sedative, stomachic, hypertensive and antispasmodic properties [Anwar, 1979; Nadkarnis,
1976]. The drug relaxes the uterus and intestine but constricts the bronchi and coronary
vessels. The fruits are used as a popular remedy for diarrhea or slight phlegmasia [Mehra,
1976]. The macerated oil shows antifungal activity [Nadkarknis, 1976]. It may be used as
tincture [Kirtikar, 1918]. Both flowers and berries are act as diuretic and can be used to treat
kidney problems and dropsy [Grieve 1892
Figure1: Photograph of whole plant of Crataegus Songorica
A B C
Figure2: Photographs of Plant with (A) fruits (B) Leaf and (C)Stem
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EXPERIMENTAL
MATERIAL METHOD
PLANT MATERIALS
Crataegus Songrica was collected from Kheri Bawli Delhi India. The plants were identified
by Prof. Dr. Anju Pal, Horticulture department, Panthnagar university, Panthnagar,
Uttarnchal, India.
PROCESSING OF PLANT MATERIAL
The plant materials were properly dried in shade for 5-6 days then dried in hot air oven at
400C after drying, the plant materials were milled to powder and passed through the sieve
(mesh size 40), this material were used for the identification of plant metabolite.
MACROSCOPICAL FEATURES
The morphological observations of roots, stems and leaves included colour, odour, taste, size,
shape, fracture, fracture surface, texture, duration and rootlets. The study of petiole included
colour, odour, taste, size, shape, fracture, fracture surface and texture., leaf study included
colour, taste, odour, size, shape, fracture, texture, margin, venation, surface, apex, duration,
type, phyllotaxis, presence and absence of petiole and stipule (Trease and Evans, 1992).
QUANTITATIVE LEAF MICROSCOPY
Quantitative leaf microscopy to determine palisade ratio, stomata number, stomata index,
vein - islet number and vein let termination number were carried out on epidermal strip.
FLUORESCENCE ANALYSIS
To check the fluorescent property of plants were powdered leaf material obtained whish is
used for to analysis under ultra and organic reagents like alcohol, 50% nitric acid and
water.16 The florescence sulphuric acid, 10% sodium hydroxide, 50% behaviour was noted
as in table.
Solubility Behaviour of Leaf Extracts Of Plants
Solubility of plant leaf extracts has been observed with different solvents.VIZ non polar,
Polar, Polar acidic and polar basic.
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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PHYSICO-CHEMICAL PARAMETERS FOR THE STANDARDISATION OF CRUDE
DRUG
Determination of foreign matter:
50 g of drug sample examined was weighed and spread out a thin layer. The foreign matter
was detected by inspection with the unaided eye. Separated and weighed it and calculated the
percent present. Drug undertaken for further study were free from moulds, insects, animal
faecal matter and other contamination such as soil, stones and extraneous material. (A.O.A.C,
2000)
Determination of moisture content (Hot Air Oven Method):
To determine the amount of moisture (water drying off from the drug) for substance
appearing to contain water as the only volatile constituent, the procedure given below, was
used. 2.78 g of drug (without preliminary drying) after accurately weighing was placed in a
tare evaporating dish. After placing the above said amount of the drugs in the tared
evaporating dish, dried at 105oC for 5 hrs, and weighed, percentage was calculated with
reference to initial weight. (BP,1980).
Calculations
W1 - W2
% Moisture = ---------------------- x100 (A.O.A.C, 2000)
Wt of the sample
Determination of ASH (PASF,1987).
Determination of Total Ash: About 2.0 g of powder drug was incinerated in a redtop silica
dish at a temperature not exceeding 4500
C. until free carbon was left, cooled and final weight
was taken. The percentage of ash calculated with reference to the air-dried drug.
Determination of Acid Insoluble Ash: The ash obtained as above method was boiled for 5 minutes
with 25 ml of dilute hydrochloric acid and collected the insoluble matter on the ash-less filter
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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paper, washed with hot water and ignited to constant weight. The percentage of acid insoluble
ash with reference to the air dried drug was calculated.
Determination of water soluble Ash: The ash was boiled for 5 minutes with 25 ml of water,
collected insoluble matter on the ash-less filter paper, washed with hot water, and ignited for
a temperature not exceeding 4500C. The weight of the insoluble matter was subtracted from
the weight of the drug ash. The difference in weight represents the water soluble ash. Finally
percentage of water-soluble ash with reference to the air dried drug was calculated.
Procedure for Determination of Ash
The ash is an analytical term used for inorganic residue that remains after the choring of
organic matter present in the food. The ash may not be the same asthe organic matter present
in the original food. There may be loss of organic matters due to the chemical reactions
among the different constituents.
Equipment and glassware
Muffle furnace, silica dish, electric balance, desiccators, Bunsen burner
Ash contents were determined by heating sample at 550 oC in muffle furnace. The procedure
is given below.
Procedure
A clean flat bottom silica dish was taken and was held in a hot burner flame for 1 minute.
After that it was transferred to a desiccator then cooled weighed (W). A suitable quantity of
food sample was weighed in china dish (W1) and heated gently on the Bunsen burner. Then
the charred mass was transferred to a muffle furnace at 550oC.
The sample was heated until all the carbons were burned. The dish (containing sample) was
placed in desiccater, cooled weighed (W2).
Weight of the empty dish = W
Weight of the empty dish + sample = W1
Weight of the empty dish + ash = W2
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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Calculations
W1 - W2
% Ash = ---------------------- x 100 (A. O. A. C, 2000)
Wt of the sample
Determination of Extractable Matter
a. Method I. Hot Extraction: About 25.0 g accurately weighed air-dried drug coarse
powder was placed in thimble and refluxed with various organic solvents hexane,
chloroform, ethyl acetate and alcohol. After recovery solvents under vacuum and
drying in desiccators, the percentage extractable matter was calculated.
b. Method II. Cold Maceration: About 2.0 g of coarsely powdered air dried
material, was accurately weighed in a glass stopper conical flask and macerated
with 100 ml of solvent for 6 hrs shaking frequently, then allowed to stand for 18
hrs, filtered rapidly taking care not to lose solvent. The extracted matter was dried
at 1050C for 6 hrs, cooled in desiccators for 30 minutes and then weighed. The
percentage extractable matter was calculated. (WHO, 1980).
Determination of Swelling Index: (WHO, 1998)
About 1.0 g fine powder accurately weighed was taken into 25 ml of glass stoppered
measuring cylinder. The internal diameter of the cylinder was about 16 mm, the length of the
graduate portion about 125 mm, marked in 0.2 ml in division from 0 to 25 ml in upward
direction. 25 ml, of water was taken and the mixture thoroughly shaken every 10 minutes for
1 hrs. Kept for 3 hrs at room temperature and the volume in ml occupied by the plant
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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material, including any sticky mucilage was measured. The mean value of the individual
determination, related to 1.0g of plant material was calculated.
Determination of Foaming Index (WHO,1998 ).
About 1.0 g a coarse powder of drug was placed into a 500 ml conical flask containing 100
ml of boiling water. The moderate boiling was maintained for 30 minutes. Cooled and filtered
into a 100 ml volumetric flask and volume was made up to the mark with distill water.
The decoction was poured into 10 stoppered test-tubes (height 16cm, diameter 16 mm) in
successive portion of 1ml, 2ml, 3ml, etc. Up to 10ml, and adjusted the volume of the liquid in
each tube with water to 10ml. The tubes were stopper and shaken them in length wise motion
for 15 seconds, two shake per second. After 15 minutes and height of the foam was
measured. The results are assessed as follows.
If the height of the foam in every tube is less than 1 cm, the foaming index is less than
100.
If the height of the foam 1 cm is measured in any tube, the volume of the plant material
decoction in this tube (a) is used to determine the index. If this tube is the first or second
tube in a series, prepare an intermediate dilution in a similar manner to obtain a more
precise result.
If the height of the foam is more than 1 cm in every tube, the foaming index is over 1000.
In this case repeat the determination using a new series of dilution of the decoction in
order to obtain a result.
Foaming index =1000\a
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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Where a = the volume in ml of the decoction used for preparing dilution in the tube where
foaming to a height of 1cm is observed.
Determination of Heavy Metals
About 0.504 g air dried material was accurately weighed and placed in the test tubes for
predigestion the test tube was contain 5ml nitric acid. It was kept as such for a day.
Now digestion was performed by adding the nitric acid and perchloric acid in the ratio of
10:4 then 5ml sample for determination was made as 3.57 nitric acid 1.42 perchloric acid.
Now it kept for heating at 170-1800C for about 4 hrs. Now sample was cool and filtered and
volume was made up to 50 ml in volumetric flask with distilled water. With the help of
instrument named as I.C.P. [O.E.S.] Model optima 5300V heavy metals was determined.
Determination of heavy metals:
Reading __
Blank = y mg / L
Yx50/0.5 = Yx100 mg/kg (ppm). Where 0.5 is wt of drug taken.
Preliminary Screening of Phytochemicals
The preliminary phytochemical studies were performed for testing the different chemical
groups present the drugs 10% (w/v) solution of extract was taken unless otherwise mentioned
in the respective individual test. The chemical group test was performed and the results are
shown in tables. General screening of various extracts of the plant material was carried out
for qualitative determination of the groups of organic compounds present in them.
Alkaliods: Test for alkaloids are following:
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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Dragendroff’s test: Dissolve a few mg of alcoholic extract of the in 5 ml of
distilled water, add 2 M hydrochloric acid until an acid reaction occurs, then add 1
ml of Dragendroff‟s reagents, orange or orange-red ppt is produced immediately.
Hager’s test: to 1 ml of alcoholic extract of the drug taken in test tube, add a few
drops of Hager‟s reagent. Formation of yellow ppt confirms the presence of
alkaloids.
Wagner’s test: Acidify 1 ml of alcoholic extract of the drug with1.5% v/v of
hydrochloric acid and add a few drops of Wagner‟s reagent. A yellow or brown
ppt is formed.
Mayer’s reagent: Add a few drops of mayer‟s reagent to 1 ml of alcoholic
extracts of the drug. White or pale yellow ppt. is formed.
Crbohydrates: Test for crbohydratesare following:
1. Anthrone test: To 2 ml of anthrone test solution, add 0.5 ml of alcoholic extracts of
the drug. A green or blue color indicates the presence of carbohydrates.
2. Benedict’s test: To 0.5 ml of alcoholic extracts of the drug add 5 ml of Benedict‟s
solution and boil for 5 mins. Formation of a brick red coloured ppt is due to presence
of carbohydrates.
3. Fehling’s test: To 2 ml of alcoholic extracts of the drug add 1 ml of the mixture of
equal parts of fehling‟s solution „A‟ and „B‟ then boil the contents of the test tube for
few mins. A red or brick red ppt is formed.
4. Molisch’s test: In test tube containing 2 ml of alcoholic extracts of the drug add 2
drops of a freshly prepared 20% alcoholic solution of β napthol mix poured 2 ml of
conc sulphuric acid so as to form a layer below the mixture. Carbohydrates, if present,
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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produce a red- violet ring, which disappears on the addition of an excess of alkali
solution.
Flavonoids: Test for flavonoids are following:
1. Shinoda’s test: In a test tube containing 0.5 ml of alcoholic extract of the drug, add 5-
10 drops of dilute hydrochloric acid followed by a small piece of magnesium. In the
presence of flavonoids a pink, reddish pink or brown colour is produced.
Triterpenoids: Test for flavonoids are following:
1. Liebermann-Burchard’s test: Add 2 ml of acetic anhydride soloution to 1 ml of
alcoholic extracts drug in chloroform followed by 1 ml of con sulphuric acid. A violet
color coloured ring is formed indicating the presence of triterpenoids.
Saponins: In a test tube containing about 5 ml of an alcoholic extracts of the drug add a drop
of sodium bicarbonate solution, shake the mixture vigorously and leave for 3 mins.
Honeycomb like froth is formed.
Steroids: Test for steroids are following:
1. Liebermann-Burchard’s test: Add 2 ml of acetic anhydride soloution to 1 ml of
alcoholic extracts of the drug in chloroform followed by 1 ml of con sulphuric acid. A
greenish colour is developed which turns to blue.
2. Salkowaski reaction:Add 1ml of conc. Sulphuric acid to 2 ml of alcoholic extracts of
the drug carefully, from the side of the test tube. A red colour is produced in the
chloroform layer.
Tannins: Test for taninsare following:
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1. To 1-2 ml of plant alcoholic extracts extract, add a few drops of 5% FeCl3 solution
was added. A green colour indicates the presence of gallotannins while brown colour
tannins.
Starch: Test for starch are following:
1. Dissolve 0.015g of iodine and 0.075g of potassium Iodide in 5 ml of distilled water
and add 2-3 ml of an alcoholic extracts of drug. A blue colour is produced.
Proteins: Test for proteins are following:
1. Biuret’s test: To 1 ml of alcoholic extracts extract of the drug add 5-8 drops of 10%
w/v sodium hydroxide solution followed by 1or 2 drops of 3% w/v copper sulphate
solution. A red or violet colour is obtained.
2. Millon’s test: Dissolve a small quantity of alcoholic extracts extract of the drug in 1
ml of distilled water and add 5-6 drops of millon‟s reagent. A white ppt is formed
which turns red on heating.
Pharmacological activity
Antibacterial Assay
Antibacterial assay was performed by agar well diffusion method (Atta-ur- Rehman et al,
2001). Methanolic extract and different portions dissolved in various organic solvent were
used in amount of three mg/mL of DMSO and pure compounds were used in dose of 1
mg/mL. Antibacterial activity was carried out against various human pathogens including
Escherichia coli (ATCC 25922), Bacillus subtilus (ATCC 6633),Shigellaflexenari (clinical
isolate), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa(ATCC 27853)
and Salmonella typhi (ATCC 19430).
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In this bio assay, three types of media are required viz. solid medium (nutrient agar), semi-
solid medium (soft agar) and liquid medium (nutrient broth).
• Solid medium (Nutrient agar) Nutrient agar
28 g Distilled water 1 L (q.s)
Nutrient agar was dissolved in distilled water and volume was made up to 1 liter. It
was then placed in auto clave at 121 0C for 15 minutes. Media was then chilled to 40
0C and
poured in sterile petri dishes and media was then left to solidify at room temperature.
• Semi solid medium (Soft agar) Soft agar 0.8 g
Distilled water 100 mL (q.s)
Soft agar was dissolved in distilled water and volume was made up to 100 mL. It was
then dispensed in 7 mL quantity to screw capped test tubes. Then placed in auto clave at 121
0C for 15 minutes and then cooled.
Liquid medium (Nutrient broth) Nutrient broth 0.8 g
Distilled water 100 mL (q.s)
Nutrient broth was dissolved in distilled water and volume was made up to 100 mL.
Prepared broth was dispensed in 3 ml quantity to screw capped test tubes, which was placed
in auto clave at 121 0C for 15 minutes and then refrigerated.The culture of bacterial
organisms was maintained on stock culture agar. A colony of bacterial culture was implanted
in nutrient bisque and incubate at 37+1 0C for 24 hours. Next day soft agar was melted and
then cooled to 40 0C and then add 100pL of bacterial culture, shake it well and then pour it
on nutrient agar containing plate. Rotate the plate to make even distribution of culture and
allow solidifying the lawn. Hole (6 mm diameter) was made in medium in each plate using a
sterile sharp tool with centers at least 24 mm apart. Samples (3 mg/ml of DMSO) were then
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added in respective well using sterilized dropping pipettes. DMSO was added other holes
and standard anti bacterial drug provided as -ve and +ve control, correspondingly. Plates
were then incubated at 37+1 0C for 24 hours. Activity was calculated by measuring diameter
of zones depicting resistance (mm). Antibacterial potential of sample was then determined as
per criteria mentioned in Table 2.5. Percent enlargement of resistance was calculated with
standard to the +ve control.
Table:2 Criteria for Determining Antibacterial Assay
Entry Diameter Activity
1 Below 9 mm No activity
2 9-12 mm Non-Significant
3 13-15 mm Low
4 16-18 mm Good
5 Above 18 mm Significant
RESULT
Macroscopic Features of Crataegus songrica fruit
The colour of Crataegus sangrica pome is reddish black with yellow pulp,
sparsely punctuate plobose, rearly ellipsid. 1.2-1. 6cm in diameter in fresh and
dark red in dry state. Odour and taste are the same in both fresh and dry forms.
Average length and diameter is 2.3 cm and 1.7 cm in fresh form and 2.0 cm and
1.5 cm in dry form, respectively. Surface is smooth and shiny in fresh form and
fluffy in dry form.
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Quantitative Leaf Microscopy
Table 4 : Quantative Leaf Microscopy of Crataegus songrica
Parameter Range
Palisade Ratio 05-10
Stomatal Number Upper surface 0
Stomatal Number Lower surface 10-15
Stomatal Index Upper surface 05
Stomatal Index Lower surface 10.44-12.32
Vein islet number 12-15
Veinlet Termination Number 6-10
Fluorescence analysis
Table 5 : Fluorescence Behaviour of Leaves of Crataegus songrica
Treatment Day light UV light
Powder as
such
Green
Fluorescent green
Powder in distilled water
Light -Green
Fluorescent yellow-Green
Powder in absolute alcohol
dark-green
Fluorescent green
Powder in 10% Nitric acid brown
Fluorescent light brown
Table 3: Macroscopical features of the fruit of Crataegus songarica.
Colour Odour Size Taste Surface Material
Ave. Length Ave. Width
Reddish Pleasant 2.5cm 1.3cm Pleasant Smooth and Shiny
Fresh
Reddish black
Pleasant 2.1cm 1.5cm Pleasant Fluffy Dried
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Powder in 50% Nitric acid
Light Yellow
Fluorescent brown
Powder in 50% Sulphuric acid
Dark Green
Fluorescent brown
Table : 6 Solubility Behaviour of Crataegus songrica of Extracts with Various
Solvents
Solvent Solubility
CPE CAC CME
NON POLAR
Heptane 1 2 0
Pet Ether 2 1 1
Cyclohexane 3 0 2
POLAR
Ethanol 1 0 3
Methanol 0 3 3
Water(dist) 3 2 3
POLAR ACIDIC
Formic acid 1 2 1
Acetic acid 2 2 0
Chloroform 0 2 3
POLAR BASIC
Pyridine 0 2 3
Dimethyl sulfoxide 2 1 0
Dimethylformamide 0 3 2
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(0): No Presence, (1): 33% Present, (2): 66% Present, (3): 100% Present. CPE:
Petroleum ether extract, CAC: Acetone extract, CME: Methanol Extract. [C =
Crataegus songrica]
Table 7 : Physico-chemical parameters of Crataegus songrica
S.No. Parameters Observations
1.
PHYSICO-CHEMICAL
Foreign Organic matter No adulterants
Ash Values (% w/w)
(a) Total Ash Value 4.23 0.01
(b) Acid Insoluble Ash 3.33 0.01
(c) Water Soluble Ash 1.33 0.01
2.
Extractive Values (% w/w)
Cold percolation method
Petroleum ether(40-60) 3.03 0.13
Methanol (95%) 2.51 0.43
Methanol (50%) 1.20 0.12
Water 1.01 1.24
Soxhlet successive extraction
method
Hexane 3.11
Chlorofrom 4.30
Ethyl acetate 0.77
Methanol (95%) 2.14
Moisture content 9.03 0.02
Pharmacological
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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Swelling Index 0.26 0.15
Foaming index 136.45
Heavy metals & Minerals ( ppm)
Cadmium 3.6
Cooper 11.2
Iron 3.10
Chromium 15.4
Manganese 2.8
Nickel 10.7
Lead 3.6
Zinc 54.6
Calcium 13.02
Potassium 4.4
Sodium 5.6
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Table 8: Phytochemical test of Crataegus songrica Leaves Extracts
Active constituents CCR CPE CAC CME
Alkalodids ++ ++ +++ ++
Flavonoids +++ +++ ++ ++
Saponins +++ ++ +++ ++
Tannins +++ ++ +++ ++
Steroids +++ ++ +++ +++
Cardiac Glycosides + ++ +++ +++
Proteins
++ ++ ++ ++
Resins _ _ _ _ _ _ _
Starch +++ +++ ++ ++
Triterpenoids -- _ _ _
Steroids _ _ _ _
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
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(-) No presence, (+) Less presence, (++) Moderate Presence, (+++) High presence,
cCR: Crude powder, CPE: Petroleum ether extract, CAC: Acetone extract,
CME: Methanol Extract, Common in CPE and CME. the Constituents can be
further isolated and purified to find its potency for biological activities. [C=
Crataegus songrica ]
ANTIBACTERIAL ASSAY
The crude methanolic extract of Crataegus songrica and its different fractions thereof
were studied against human pathogens Escherichia coli ATCC 25922, Bacillus
subtilis ATCC 6633, Shigella flexeneri (clinical isolate), Staphylococcus aureus
ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Salmonella typhi ATCC
19430. They were remained on agar skeat 4o C. The strains were make actived at 37
o
C for twenty four hours on nutrient agar (NA) or Sabouraud glucose agar (SGA) for
bacteria, before screening.
Crude extract of Crataegus songrica and various segments revealed region of
inhibition in millimeters alongside various bacterial strain percent comparisons to
standard drugs i.e. Imipenum in table. The dose (3 mg/ml) was given in single
concentration. Crude methanolic extract (SC-1), n-hexane fraction (SC-2) and ethyl
acetate (SC-4) showed well to significant activity against Escherichia coli and the
zone of inhibition were 12 mm, 16 mm and 19 mm respectively. (Table ) and showed
no inhibitory activity against other test organisms. In other fractions, varied inhibitory
activity was observed against different bacterial strains. The n- hexane showed the
zone of inhibition only against Escherichia coli, with diameter of 16 mm. Chloroform
fraction (SC-3) inhibited the activity of Bacillus subtilis and Shigella flexenari 14 mm
and 18 mm each. While fraction ethyl acetate (SC-4) illustrated the inhibitory effect
only against Escherichia coli and Bacillus subtilis inhibited growth 19 mm and 15
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mm respectively. Butanol fraction (SC-5) inhibited the growth of Bacillus subtilis and
S. aureus, Pseudomonas aeruginosa with the zone of inhibition 19 mm, 11 and
12mm respectively. While in case of aqueous fraction (SC-6), inhibitory activity was
observed against growth of Shigella flexenari and Pseudomonas aeruginosa by 11
mm and 10 mm each.. Crude extract of plant exhibited potential antibacterial activity
against E. coli
Table 9: Results of antibacterial assay of crude extract and fractions
Entry Sample E. coli B. subtilus S. flexenari S. aureus P. aeruginosa S. typhi
1 2 1 2 1 2 1 2 1 2 1 2
1 SC-1 12 58 9 39 15 53 - - - - - -
2 SC-2 16 58 - - - - - - - - - -
3 SC-3 - - 14 52 18 57 - - - - - -
4 SC-4 19 70 15 56 - - - - - - - -
5 SC5 - - 19 70 - - 11 40 12 - - -
6 SC-6 - - - - 11 52 - - 10 39 - -
7 STD 24 - 23 - 28 - 27 - 20 - 26 -
E. coli:Escherichia coli S. aureus: Staphylococcus
aureus
B. subtilus:Bacillus subtilus P. : Pseudomonas aeruginosa
S. flexenari: Shigella flexenari S. typhi : Salmonella typhi
SC-1: Crude Methanolic extract SC-5: n-BuOH fraction
SC-2: n-hexane fraction SC-6: H2O fraction
SC-3: CHCl3 fraction STD: Imipenum.
SC-4: EtOAc fraction
1) Zone of inhibition (mm) 2) Percent inhibition to standard drug
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REFERENCE
1.Kokate, C. K; Purohit, A. P and Gokhal, S. B., 2006. Pharmacognosy 30th
ed. Nirali
Prakashan, Pune, India.
2.Dev, S., 1988. Ayurveda and modern drug development. Proc Indian NatI Sci Acad
Part-A Phys Sci, Vol. 54A: pp 12-42
3.Sharma, S. P, Charaka Samhita., 1981. Vol. 1,-I. Chaukhambha. Oriental, Varanasi,
India.
4.Krishnamurthy, K. H., 1999. Wealth of Susruta Coimbatore, lnternational Institute of
Ayurveda, Coimbatore, Kerala, India. pp. 1466-1469.
5..Garde, G.K., 1954. Sartha Vagbhatta Ashtanga Hridaya. Aryabhushana Mudranalaya,
Pune, India.
6.Hartzell J. F; Zysk, K. G., 1995. Health, science, and the spirit: Veda and Ayurveda in
th
7.Western world. Journal of Alternative Complementary Med, Vol. 1: pp. 297-301.
8.Anwar., A.K, Ashfaq., M, Ali, M.N. Pharmacognostic studies of slected plants of
Pakistan Forest Institute, Peshawar. 6, 7 (1979).
9.Nadkarni,s, K. M. “Indian Materia Medica”, Vol.I&II, Popular Prakashan Private
10. Limited, Bombay (1976).
11. Shinwari, Z.K. and Rehman.Takashi Watanabe, Takafumi Yoshikawa. A pictorial
12. guide to medicinal plants of Pakistan.. 231. (2006).
13. Hobb, C., and Foster, S. Hawthorn. Herbal Gram, 22, 19-33.(1990).
14. Brown , D. Encyclopedia of herbs and their uses. Dortling Kinderley
Publishers.(1995).
15. Kiritikar, K. R and Basu, B. D. “Indian Medicinal Plants”. Part-I, Indian Press,
Allahabad (1918).
16. Mehra, P. N and Puri, H. S. Res. Bull. Punjab Univ., 19(3-4) 439 (1968).
International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)
Volume III, Issue 3 March 2017
All rights are reserved 79
17. Trease, G. E and W. C. Evans. Pharmacognosy. 1992). 13th ed. English Language
Book, Society Baillere Tindall, Oxford University Press. 17, pp.417.
18. AOAC. Association of Official Analytical Chemists, Gaithersburg, MD, USA.
17thedition.
19. (2000).
20. BP, British Pharmacopoeia. 1980. Her Majesty's Stationery Office (HMSO), London,
Great
21. Britain.
22. PSAF, Pharmacopoeial Standards for Ayurvedic Formulations., 1987. Central council
23. for Research in Ayurveda and Siddha (CCRAS). Ministry of Health and Family
Welfare,
24. Govt of India, New Delhi.
25. WHO. 1998. Quality control methods for medicinal plant material, World Health
26. Organization Geneva.