phytochemical evalution and in-vitro anthelmintic activity

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




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].




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].




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].




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.




(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




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




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




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



50 mg/ml 21 min,48 sec ±14 24 min,48 sec ±14

Aqueous extract



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




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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phytochemical evalution and in-vitro anthelmintic activity

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