Abinash kumar sahu, et al., 2020/ Phytochemical Evalution and In-vitro Anthelmintic
International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 5 (4) 1
RESEARCH ARTICLE
International Research Journal of Pharmaceutical and Biosciences
Pri -ISSN: 2394 - 5826 http://www.irjpbs.com e-ISSN: 2394 - 5834
Phytochemical Evalution and In-vitro Anthelmintic Activity of Oldenlandia corymbosa Abinash Kumar Sahu*, Amiya Ranjan Padhan, Nishigandha Sa, Anjan Kumar Padhan
The Pharmaceutical College, Tingipali, Barpali, Bargarh, B.P.U.T Odisha.
Article info Abstract
Article history:
Received 15 JULY 2020
Accepted 26 JULY 2020
*Corresponding author: [email protected]
Copyright 2020 irjpbs
ABSTRACT: The plant Oldenlandia corymbosa belongs to family “Rubiaceae”. The plant is used as anthelmintic, antirheumatic, depurative, diaphoretic, digestive, diuretic, febrifuge, pectoral , stomachic, treat jaundice and other liver conditions and to treat viral infections, cancer, acne, boils, appendicitis, hepatitis, eye problems and bleeding etc Aim; The aim of the study is to investigate Phytochemical screening of the Petroleum ether and Aqueous extract of Oldenlandia corymbosa and the presence of different secondary metabolites responsible for the therapeutic values of the drug and also to find out the anthelmintic activity study by in-vitro test species Pheretima posthuma responded towards the plant extracts by showing the sign of paralysis and death finally Results; The % of yield of petroleum ether extract and Aqueous extract were found to 10.92% w/w and 5.45%w/w. In fluorescence analysis the powder drug was treated with different reagent which showed different colours in U.V light. Conclusion: The % yields of both extracts were found to 10.92% w/w and 5.45%w/w. The preliminary phytochemical screening showed the presence of alkaloids, carbohydrate, saponins, Proteins & amino acid, Phenolic & flavonoids compounds and shows the absence of Phytosterol. In fluorescence analysis the powder drug was treated with different reagent which showed different colours in U.V light. The data revealed that the aqueous extract has a better wormicidal effect than Petroleum Ether extract. KEY WORD: Oldenlandia corymbosa, medicinal use, phytochemical screening, Phytochemical, Albendazole, Anthelmintic Activity, Pheretima posthuma.
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INTRODUCTION
Plants have been used in medicines since time immemorial. India has a rich heritage of using
medicinal plants in traditional medicines, as in the Ayurveda, Siddha and Unani systems
besides folklore practices. The earliest inscription of the medicinal uses of plants is found in
the “Rigveda”, which is one of the oldest repositories of human knowledge. Fairly
comprehensive information on the curative properties of some herbs has been found
recorded in “Charak Samhita” and “Sushruta Samhita”. The plant kingdom is a virtual
goldmine of biologically active compounds and it is estimated that only 10-15% of existing
species of higher plants have been surveyed. Many plants have been successfully used in the
treatment of various diseases. The ancient record is evidencing their use by Indian, Chinese,
Egyptian, Greek, Roman and Syrian dates back to about 5000 years. In India, around 20,000
medicinal plant species have been recorded recently, but more than 500 traditional
communities use about 800 plant species for curing different diseases [1]. Currently, 80% of
the world population depends on plant-derived medicine for the first line of primary health
care for human alleviation.
TAXONOMY AND ETHNO MEDICINE OF OLDENLANDIA CORYMBOSA
Scientific Classification [2]
Rank Scientific Name
Kingdom :Plantae
Subkingdom :Tracheobionta
Superdivision :Spermatophyta
Phylum :Angiosperms
Division :Magnoliophyta
Class :Magnoliopsida
Subclass :Asteridae
Order :Rubiales
Family :Rubiaceae
Subfamily :Rubioideae
Genus :Oldenlandia L.
Species :Oldenlandia corymbosa L.
Synonyms :Hedyotis corymbosa, Hedyotis biflora, Gerontogea
corymbosa, Gerontogea biflora, Gerontogea herbacea.
In different parts of India Oldenlandia corymbosa is known by different names[3]
Sanskrit : Parpata, Parpataka
English : Diamond flower
Hindi : Daman pappar, Pitpapra
Malayalam : Parpatakapullu, Parpatakam
Gujarati : Parpat, khet-papra
Marathi : Papti, Phapti, khet-papda, paripat
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Tamil : Parpatagam, kattucayaver, pappanpuntu
Telugu : Verrinella- vemu
Kannada : Parpatahullu, Kallasabatrasige
Bengali : Khet-papra
GEOGRAPHICAL DISTRIBUTION [4]
Oldenlandia corymbosa is native to India, but also found throughout Malaysia, Sri Lanka,
tropical East Asia to Java, Africa and Phillipines. It is a weed habitually growing in sandy
lands and waste places. It is dispersed all over India, Baluchistan, Ceylon, tropical and
subtropical Asia and Africa.
PLANT PROFILE: [5]
Oldenlandia corymbosa is known as diamond flower which is an annual, terrestrial,
dichotomous, slender herb with ascending or erect stems up to 50 cm long ridged, glabrous
or scabridulous or pubescent on the ribs which are 4-angled. Linear-oblong or narrowly
elliptic, almost stalkless leaves are 1-3 to 2 cm long and by 0.8 -3 mm wide, rough on margins.
The lower leaves are often broader than upper ones, linear, acute, glabrous, usually with
recurved margins. Midrib of the leaf is prominently visible. Flowers are borne in 2-8
flowered cymes in leaf axils. Flowers are white in pairs or in threes or faintly pinkish-
purplish, on slender stalks 4-8 mm long and usually on solitary axillary peduncles longer
than the calyx. Flower tube is about 2 mm long, with 4 petals. Stamens are inserted just above
the base of the tube. Capsule is about 2 x 2 mm, flattened at apex, slightly laterally
compressed. Fruits are loculicidal capsules, globose and the seeds are minute, pale brown,
angular, testa teticulate. These capsules are about 2 x 2 mm, exalate, flattened at apex,
slightly laterally compressed Diamond Flower is found in found all over the tropical world.
It is also found in the Himalayas.
Figure.1– Oldenlandia corymbosa herb
Habit: This Oldenlandia corymbosa herb is sparsely to very densely branched near the base
and mainly found in grassland, crop field, mountain scrub, shallow soil on rocks, lawn,
garden, waste places, roadsides, cultivated and disturbed ground (Fig. 1), at elevations from
sea level to 2000 meters [6].
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Macroscopical characters [7, 8, 9, 10]
Leaves: leaves are subsessile, linear-lanceolate, acute, recurved and hairy margins,
stipulate, stipules membranous, truncate with a few bristles. Leaves are opposite, linear
lanceolate, 1-3 cm long and 2-4 cm wide. They are sessile, acute at the apex, entire at the
margins, smooth, pale green beneath and dark green above. leaf is entire, secondary veins
are sometimes or not at all visible; stipules are fused to petiole bases.
Stem: Stem of the plant is 4-angled to flat glabrous and angles are thick to wing. Stem is
woody at the base, more or less angled, slender and glabrous.
Inflorescence: Axillary solitary. Is a corymbose-umbel with 2-4 flowers on a peduncle
up t0 2 cm long, occurring in leaf axils all along the stem.
Flowers: The flowers are White, small peduncles, pedicels 2-3 on the top, bract single
below the pedicels. Calyx teeth 4, triangular, corolla lobes 4, acute, valvate. Stamens 4, in
the throat of the corolla. Ovary 2-celled, ovules many, stigmas 2, linear. white, 2cm in
diameter on pedicels up to 1 cm long.
Calyx: Calyx-tube ellipsoid, 0.7–1 mm. long; lobes triangular, 0.5–1.8 mm. long, setulose
on the margins.
Corolla: Corolla white or tinged blue, pink or purple or with 2 pink stripes on each lobe;
tube 0.6–1 mm. long; lobes ovate to oblong, 0.5–1.2 mm. long.
Capsule: Capsule ovoid, 1.2–2.2 mm. tall, (1–)1.8–2.8 mm. wide, the beak scarcely raised.
globose, these capsules are with two carpels.
Flowering and Fruiting Time: August - October
Seed: Seeds are, smooth, numerous minutes, round and brownish pale brown, ellipsoid
or very obtusely depressed conic, ± 0.3 mm. long, reticulate.
Fruit: Fruit capsular, sub-globose, ovoid, 1.2-2×1.2-2.2 mm size, dehiscent through flat
to broadly rounded apex, beak when present to 0.5 mm, peduncles and pedicels usually
extending promptly and prominently as the fruit develops.
CHEMICAL CONSTITUENTS:
The chemical constituents reported in different parts of Oldenlandia corymbosa are
mentioned beneath.
1) Different phytochemical studies show the presence of proteins, carbohydrates,
phenols, tannins, flavanoids, saponins, steroids, terpenoids, coumarin and glycosides.
Some of the isolated compounds from whole plants are Geniposide, iridoid glycosides,
6 alpha – hydroxygeniposide, scandoside methyl ester (6 beta - hydroxygeniposide),
10-o-benzoylscandoside methyl ester, asperulosidic acid, asperuloside,
deacteylasperuloside, 10-o-p-hydroxy benzoylscandoside methyl ester, rutin and (+)-
lyoniresinol-3-alpha -o-beta glucopyranoside [11,12,13].
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2) The plant also contains ursolic acid, oleanolic acid and γ-sitosterol. The air-dried plant
contains 0.12% of alkaloids – bifloron and biflorin, these two alkaloids are interconvertible.
It also contains 13.55% of inorganic ash, which is mainly responsible for its cooling effect
[14].
3) An aqueous extract of the plant yielded a polysaccharide, composed of rhamnose,
arabinose, zylose, mannose, galactose and glucose.
4) The methanol extracts of Oldenlandia corymbosa showed the presence of flavonols such
as Quercetin, 3‟-Methoxy quercetin and 3‟, 4‟-Dimethoxy quercetin. Phenolic acids like
vanillic, syringic acid, melilotic acid, p-hydroxy benzoic, p-coumaric, ferulic and caffeic acids
are also present. Anthocyanidins like cyanidin and pelargonidin are present. Irridoids and
alkaloids are also present [15].
The freshly prepared crude extract was qualitatively tested for the identification of chemical
constituents, such as, alkaloids, flavonoids, steroids, glycosides, saponins, terpenoids, gums
and tannins. [16]
TRADITIONAL USES [17, 18, 19, 20]
1. The leaves are pounded, soaked in warm water and the liquid drunk to treat stomach
disorders
2. They are used externally as a poultice to treat sores and sore eyes
3. The roots are reported to have vermifuge properties. They are often used as a
tincture.
4. The roots yield a green dye known as 'gerancine' after treatment with a mordant.
5. The entire plant is used in decoction as an anthelmintic, antirheumatic, depurative,
diaphoretic, digestive, diuretic, febrifuge, pectoral and stomachic
6. It is also used to treat jaundice and other liver conditions.
7. In Chinese medicine it is used to treat viral infections, cancer, acne, boils, appendicitis,
hepatitis, eye problems and bleeding
8. The entire plant is used in helminthiasis, leprosy, skin diseases, cough, bronchitis,
necrosis and nerve depression
9. In Africa it is used to facilitate child birthatic, depurative, diaphoretic, digestive,
diuretic, febrifuge, pectoral and stomachic.
The plant is known to clear heat and toxins, activate blood circulation, promote
diuresis and relieve strangury. It is also known to act on lymphosarcoma and carcinoma of
the liver and larynx. It is also active against appendicitis, hepatitis, pneumonia, cholecystesis,
urinary infection, cellulites and snake bite. Chinese folk medicine describes the plant to treat
skin sores, ulcers, sore throat, bronchitis, gynecologic infections and pelvic inflammatory
diseases [21, 22, 23, 24].
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A scrutiny of literature revealed some notable pharmacological activities of the plant such as
hepatoprotective, cytotoxic anti-oxidant, oxytocic and anti-malarial activity [25]. The plant
is widely used in treating various diseases associated with kidney.
MATERIALS AND METHODS
The following drugs and chemicals were used for the different experimental study.
The Mayer's, Hager’s, Barfoed’s, Benedict’s and millon’s reagent were purchased from S.D.
Fine Chemical, Mumbai. The solvents petroleum ether, Chloroform was purchased from Hi
Media Laboratories Pvt. Ltd., Mumbai. Methanol and Petroleum ether was purchased from
Qualigens chemicals. Mumbai. And all others chemicals, solvents and reagents were of
analytical grade and procured from authorized dealer.
Plants collection, Identification and processing:
The stems of Oldenlandia corymbose were collected from adjoining area of Barpali
(Dist-Bargarh, Orissa) in the month of October 2019. The plant was identified by and
authenticated By Prof. (Dr.) Santosh Kumar Dash, Retired Professor and H.O.D., P.G Dept. of
Biosciences, C.P.S, Mohuda, Berhampur, Ganjam, Odisha. on dated 02-10-2019. The plant
was washed properly with water to remove the mud or dust, and then it was dried in sun
light for 1 hour and kept in shade dried and powdered by the help of mechanical process.
The coarse powder has stored in air tight container for further studies.
PREPARATION OF EXTRACTS
Preparation of the extracts of Oldenlandia corymbosa powdered of entire plant by using
following solvents:
(a) Pet ether Extract
(b) Aqueous Extract
(a) Pet ether Extract
The shade dried course powder of the entire plant (200 gm) was packed well in soxhlet
apparatus and was subjected for continuous hot extraction with Pet ether extract. The
extract was filtered while hot and the resultant extract was distilled in vacuum under
reduced pressure in order to remove the solvent completely. Dried and kept in a desiccator
till experimentation. Obtained extract (dark wine red) was weighed and percentage yield
was calculated in terms of air-dried powdered crude material.
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(b) Aqueous extract
The shade dried course powder of the entire plant (200 gm) was packed well in soxhlet
apparatus and was subjected to continuous hot extraction with distilled water until the
completion of extraction. The extract was filtered while hot and the resultant extract was
distilled in vacuum under reduced pressure in order to remove the distilled water
completely. It was finally dried and kept in a desiccator till experimentation. Obtained
extract (dark reddish brown) was weighed and percentage yield was calculated in terms of
air-dried powdered crude material.
The yield and % yield of both Petroleum ether and aqueous extracts of powdered of entire
plant Oldenlandia corymbosa were reported below.
Table-1 Extraction values of Hydro-alcoholic and aqueous extracts of Oldenlandia
corymbosa
PRELIMINARY PHYTOCHEMICAL SCREENING:
The plant may be considered as a biosynthetic laboratory, not only for the chemical
compounds such as Carbohydrates, Protein and Lipids that are utilized as food by human
being but also for a multitude of compounds like Glycosides, Alkaloids, Volatile oils, Tannins
etc., that exerts a physiological effect. The compounds that are responsible for therapeutic
effect are usually the secondary metabolites. A systemic study of a crude drug embraces
through consideration of both primary and secondary metabolites derived as a result of
plant metabolism. The plant material may be subjected to preliminary phytochemical
screening for the detection of various plant constituents. The different qualitative chemical
tests can be performed for establishing profile of given extract for its chemical composition.
The following tests may be performed on extracts to detect various phytoconstituents
present in them [25].
Methods:
Qualitative phytochemical studies of extract will be studied for the presence of different
secondary metabolites responsible for the therapeutic values as per the standard procedures
mentioned in Pharmacopoeia [26,27]. The solvent free extract of about 50 mg was stirred
with few ml of dilute hydrochloric acid filtered. The filtrate was tested carefully for different
constituents.
Sl. No. Extracts % Yield (w/w) Colour of extract
1. Petroleum ether 10.92% Dark wine red
2. Aqueous 5.45% Dark reddish brown
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Table-2Qualitative Phytochemical analysis of Petroleum ether and aqueous extracts
of Oldenlandia corymbosa.
Phytochemical test Petroleum ether
Extract
Aqueous Extract
Alkaloid test
Mayer’s test Present Present
Wagner’s test Present Absent
Hager’s test Present Present
Dragendorff’s test Present Present
Carbohydrates
Molish’s test Present Present
Fehling’s test Present Absent
Barfoid’s test Absent Absent
Benidict’s test Present Absent
Borntrager’s test Present Present
Saponins
Foam test Absent Present
Proteins & amino acid
Millon’s test Present Present
Biuret’s test Present Absent
Ninhydrin test Present Present
Phenolic compounds & flavonoids
Ferric chloride test Absent Absent
Lead acetate test Present Absent
Alkaline test Present Positive
Phytosterol :
Libermann-Burchard’s
test
Absent Absent
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Table-3 Powder analysis with chemical reagent
Table-4Fluorescence analysis of powder drug
Reagent Colour
observed
(naked eye)
Colour
observed (U.V
short wave
length)
Colour
observed (U.V
long wave
length)
Powder as such Light Brown
Light Brown
Dark brown
Powder + 1N NaOH
in methanol
Yellowish
brown
Light green Dark brown
Powder + NaOH in water Light brown Light brown Dark brown
Powder + 50% HCl yellowish
brown
Light green Black
Powder +50% H2SO4 Light brown Light green Black
Powder +50% HNO3 Light brown Green Dark brown
Powder + petroleum ether Light brown Light brown Dark brown
Powder + chloroform brown Green Dark brown
Powder + picric acid yellow Green Black
Powder + 5% FeCl3 Yellow green Green Black
Powder + 5% iodine solution Dark green Dark green Black
Powder + methanol Light brown Dark brown Dark brown
Powder + (HNO3 + NH3) Light brown Yellow green Dark brown
Reagents Colour of powder
Powder as such Light Brown
Powder + conc. HCI Light yellow
Powder + conc. HNO3 Yellowish brown
Powder + conc. H2SO4 Dark brown
Powder + glacial acetic acid Brown
Powder + dil. HCI Light brown
Powder + NaOH sol. Light brown
Powder + FeCl3 Yellowish green
Powder + picric acid Yellow
Powder + ammonia Light brown
Powder +Iodine Brown
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DETERMINATION OF ANTHELMINTIC ACTIVITY [ 28] :
The anthelmintic study was done by using one in-vitro species adult earthworms Pheretima
posthuma. Earthworms were collected near the swampy water in our locality. The average
size of the round worm was 5-7 cm; average size of the earthworm was 8-9 cm. These
earthworms were identified and services of veterinary practioner were utilized to confirm
the identity of worms. The suspensions of various extracts were prepared in 2% gum acacia
solution to obtain 1, 2.5 and 5% concentrations. Solutions of similar concentrations of the
standard drug albendazole were also prepared in distilled water.
Two ml of each concentration of various extracts of Oldenlandia corymbosa and
standard drug albendazole were diluted to 10 ml separately with normal saline and poured
in petridishes. 2ml of 2% gum acacia solution was diluted to 10ml with normal saline to serve
as control. Six earthworms of nearly equal size were placed in each Petridis at room
temperature.
Table 5: Anthelmintic effect of oldenlandia corymbose extracts.
Group Concentration of Extract (%)
Time in minutes (Mean ± SEM)
Paralysis time (Min)
Death time (Min)
Albendazole (standard)
10 mg/ml 14min,18 sec ±17 18min,18 sec ±42
30 mg/ml 10min,21 sec ±12 14 min,12 sec ±10
50 mg/ml 8 min,12 sec ±14 12min,40 sec ±11
Petroleum ether extract
15 mg/ml 27min,16 sec ±17 32min,15 sec ±48
30 mg/ml 23min,26 sec ±12 30 min,26 sec ±12
50 mg/ml 21 min,48 sec ±14 24 min,48 sec ±14
Aqueous extract
15 mg/ml 12min,19 sec ±17 20min,15 sec ±48
30 mg/ml 12min,26 sec ±12 16 min,26 sec ±12
50 mg/ml 10 min,48 sec ±14 13min,14 sec ±10
Control - - -
Results are expressed as mean ± SEM from six observations, Control worms were alive up to 24
hrs. of observation, N/A= No Activity shown within 24 hours.
Time was recorded at the time of releasing the earthworms to each concentration.
The time taken (minutes) for the complete paralysis and death were recorded. The mean
paralysis time for each sample was recorded. The anthelmintic activity was evaluated on
adult Indian earthworm Pheritima posthuma due to its anatomical and physiological
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resemblance with the intestinal round worm parasites of human beings. Paralysis was said
to occur when the worms did not revive even in normal saline. Death was concluded when
the worms lost their motility followed by fading away of their body colour.
RESULTS AND DISCUSSIONS
The percentage yield of Petroleum ether and aqueous extract were found to 10.92%w/w and
5.45%w/w (Table-1). The preliminary phytochemical screening on the leaf extract was
carried out by subjecting the different extracts to qualitative test for the identification of
various plant constituents. It showed the presence of alkaloids, glycosides, saponins,
anthraquinone, terpenoids, phlobatanin like compounds (Table-2) but do not shows the
presence of carbohydrate in Petroleum ether, phenolic and flavonoid. The Powder analysis
and fluorescence was observed in ultra-short, ultra-long and visible. The results were shown
in (Table-3 & 4).
The results (Table-5) depict the time taken for paralysis and death of earthworms after the
treatment with the test extracts at the selected concentrations. The data revealed that the
aqueous extract has a better wormicidal effect than Petroleum ether extract with compared
with the standard drug Albendazole. Further study is required to find out the novel
phytoconstituents responsible for anthelmintic action against various helminthes.
ACKNOWLEDGEMENTS:
The authors sincerely thanks to the Principal and Management of The Pharmaceutical College, Barpali, Bargarh for providing all the facilities to carry out the study.
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