Phytopharmacological Study of Areca catechu leaf

 

Nishant. M. Patel1*, H. P. Suryawanshi, S. P. Pawar

P.S.G.V.P. M’S College of Pharmacy, Shahada, (MS), India

*Corresponding Author E-mail: nishantpatel264@gmail.com

 

ABSTRACT:

Areca catechu (Arecaceae) is an erect unbranched palm tree, growing mainly in south and Southeast Asian countries. It is commonly used in day to day life. In present investigation an attempt has been made for the pharmacognostic standardization and phytochemical evaluation of Areca catechu leaves. The pharmacognostic evaluation comprises of detailed macroscopy, powdered microscopy, quantitative microscopy and physical constants such as ash and extractive values. The leaves extract were subjected to preliminary phytochemical screening. The data obtained in present study will serve as valuable tool for identification, authentication and detection of adulterants standardization and quality control of the drug. In this research work the anthelmintic property of leaves extract of Areca catechu belonging to family Arecaceae was studied for anthelmintic activity against Pheritima Posthuma (Indian earthworm). The leaves powder of Areca catechu was subjected to successive extraction by maceration using solvents like Acetone + Aqueous, Pet. Ether and Alcohol. Four concentrations (25, 50, 75 and 100 mg / ml) of different extract of leaves were studied in a bioassay, which involved the determination of time of paralysis and time of death of the worms. 100 mg/ml conc. of aqueous + acetone extract of leaves of Areca catechu reveal considerable anthelmintic activity as compared to other three conc. and piperazine citrate (10 mg/ml). Piperazine citrate and saline water were including in the assay as standard reference drug and control, respectively.

 

KEYWORDS: Areca catechu, Anthelmintic activity, Phytochemistry.

 

 


INTRODUCTION:

Areca catechu of family Arecaceae is an erect unbranched palm reaching heights of 12-30 m, depending upon the environmental conditions also known as supari (Hindi). The adult palm has 7-12 open leaves, each with a sheath, a rachis and leaflets. The leaf stalk extends as the midrib until the end of the leaf and ends as leaflets. Male flowers very numerous, sessile, without bracts; calyx 1-leaved, small, 3-cornered, 3-parted; petals 3, oblong, rigid striated stamens 6, anthers sagittate.

 

Female flowers solitary or 2 or 3 at or near the base of each ramification of the spadix, sessile, without bracts; sepals permanent staminodes 6, connate, styles scarcely any; stigmas 3, short, triangular. Fruit a monocular, one-seeded berry, 3.8-5 cm long, smooth orange or scarlet when ripe, with a fibrous outer layer.(1)

 

They have various utilities of areca catechu like anthelmentic, Antibacterial, Anti-inflammatory, oxytocic clastogenic, Anti-hypertensive, Anti-covulsant, Anti-venom activity.

 

The literature has revealed that seeds and leaves of plant contain about six alkaloids of which four (Arecoline, Arecaidine, Guvacine and Guvacolin) has been conclusively identified in biochemical studies. Polyphenols (flavonols and tannins) are responsible for the astringent taste of nut.(2)

 

MATERIAL AND METHODS:

Procurement of Plant Material:

The leaves of Areca catechu have been collected from the local region of Shahada (M. S). The plant is authenticated by Dr. Santosh Tayade, Dept. of Botany, Art’s Science and Commerce College, Lonkheda, Shahada, Dist-Nandurbar (M.S). The voucher specimen has been preserved in the laboratory for future reference in the Dept. of Pharmacognosy and Phytochemistry, College of Pharmacy, Shahada.

 

The collected plant material was air dried and used for the study of macroscopic and microscopic characters. Finally dried leaves were subjected to size reduction to get coarse powder and then passed through sieve no 40 to get uniform powder. Then uniform powder was subjected for the determination of ash values, extractive values, and loss on drying and phytochemical constituents.

 

I] PHARMACOGNOSTIC EVALUATION:

1) Organoleptic Evaluation(3)

In organoleptic evaluation, various sensory parameters of the material such as color, odour, taste, shape, margin, venation, arrangement, type and texture of the leaves were recorded and shown in table no.2.

 

2) Microscopical Evaluation:

i) Transverse Section of Areca catechu leaves:

The transverse section of the Areca catechu leaves was taken to observe microscopic characteristics like upper epidermis, phloem, xylem, collenchyma, spongy parenchyma, lower epidermis etc. The characteristic are shown in microscopic pictures as fig. no.1.

 

ii) Powder Analysis:

To a little quantity of powder taken onto a microscopic slide, 1–2 drops of 0.1% phluroglucinol solution and a drop of concentrated hydrochloric acid were added, covered with a cover slip and observed under microscope with 10 × 10 magnification. The characteristic features of the powder viz., epidermal cells, vessels, xylem fibres etc. were recorded using standard techniques. Xylem vessels and fibers appear pink in colour.

 

II) Physical Evaluation:

In physical evaluation, ash values viz., total ash, acid insoluble ash and water soluble ash, and extractive values viz., water soluble extractive and pet ether extractive values were determined.

 

1) Determination of ash Values(4)

a)    Determination of Total Ash Value:

Accurately weighed (2 g) of air-dried Areca catechu leaves powder was taken in a silica dish and incinerated at a temperature not exceeding 450°C until free from carbon. The resultant ash was cooled and weighed. The percentage of ash was calculated with reference to the air dried drug. The results were given below.

 

b)    Determination of Acid Insoluble Ash:

The total ash obtained from (2g) of Areca catechu leaves powder was boiled for 5 minutes with 25 ml of dilute HCL and the insoluble matter was collected on an ash less filter paper. It was washed with hot water, ignited and weighed. The percentage of acid insoluble ash was calculated with reference to the air dried drug. The result was given below.

 

c)     Determination of Water Soluble Ash:

The total ash obtained from (2 g) Areca catechu leaves powder was boiled for 5 minutes with 25 ml of water, the insoluble matter was collected on an ash less filter paper, washed with hot water, and ignited for 15 min at a temperature not exceeding 450°C. The weight of insoluble matter was subtracted from the weight of the ash, the difference in weight represent the water-soluble ash was calculated with reference to the air-dried drug. The results were given below.

 

2) EXTRACTIVE VALUES(5):

a)    Petroleum ether (40-60°C) soluble extractive value:

Accurately weighed (5 g) of leaves powder of Areca catechu was macerated with 100 ml of Petroleum ether in a closed flask, shaking frequently during the first 6 hours and allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precaution against loss of Petroleum ether. Evaporated 25ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105°C and weighed. Percentage Petroleum ether soluble extractive was calculated with reference to the leaves. The results of extractive values are given below.

 

b)    Water-soluble extractive value:

Accurately weighed (5 g) of leaves powder of Areca catechu was added to 50 ml boiled water at 80°C in a stoppered flask. It was then shaken well and allowed to stand for 10 min so as to cool it and filtered. 5ml of filtrate was transferred to an evaporating dish, which was 7.5 cm in diameter; the solvent was evaporated on water bath, allowed to dry for 30 minutes, finally dried in an oven for 2 hrs. At 100°C and residue was weighed. Percentage of water soluble extractive value was calculated with reference to the air dried drug. The results are given below.

 

3) LOSS ON DRYING(6):

Accurately weighed (2 g) quantity of leaves powder of Areca catechuwas taken in tarred glass bottle and initial weight was taken. The sample was heated at 105°C in an oven and weighed. This procedure was repeated until a constant weight was obtained. The moisture content of the sample was calculated with reference to air-dried drug and results are given below.

III) Preliminary Phytochemical Screening(7):

The leaves powder are subjected to successive extraction by Maceration by using water + acetone, the extracts where evaporated to dryness. The dried extracts were weighed.

 

Preliminary Phytochemical Screening:

The percentage yield of leaves of Areca catechu after maceration was found to be max of 7.46 gm (Aqueous + acetone extract).

 

The extract were used for preliminary phytochemical with a chemical tests viz., Molisch’s, fehling’s, Benedict’s and Barfoed’s tests for carbohydrates; Ninhydrin’s test for amino acids; Salkowski and Libermann-Burchard’s reaction for steroids; Foam test for saponin glycosides; Dragendorff’s, Mayer’s, Hager’s and Wagner’s tests for alkaloids; and ferric chloride, lead acetate, potassium dichromate, and dilute iodine tests for tannins, shinoda test for flavonoids The results are given below in table no.1.

 

Table No. 1: Preliminary phytochemical screening

Phytoconstituents

Aqueous + Acetone Extract

Alkaloid

+

Carbohydrate

+

Saponins

+

Tannins

+

Steroids

+

Glycoside

+

Amino acid

-

Flavonoids

+

 

Phytoconstituents present in extract of leaves of Areca catechu:

In the present investigation all the extracts of plant was analyzed for the presence of carbohydrates, alkaloids, amino acids, tannins and saponins by using standard procedures. The preliminary phytochemical investigation showed the presence of carbohydrate, alkaloids, flavonoids, steroids, saponins and tannins as shown in table no.1.

 

IV) Pharmacological Evaluation:

1) ANTHELMINTIC ACTIVITY:

The anthelmintic assay was carried as per the method of Ajaiyeoba et.al, with necessary modification.(8) The assay was performed on adult Indian earthworm Pheritima Posthuma, due to its anatomical and physiological resemble with the intestinal round worm parasite of human being.(9,10) Because of easy availability, earthworm has been used widely for initial evaluation of anthelmintic compound in vitro.(11) 25 ml of formulation containing different concentration of crude drug aqueous + acetone extract (25, 50, 75, 100 mg/ml in distilled water) were prepared and 3 worms of same type were placed in it. Time for paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously. Time for death of worms were recorded after ascertaining that worms neither move when shaken vigorously nor when dipped in warm water (50°C). Piperazine citrate (10 mg/ml) was used as reference standard while saline water as control.

 

MATERIALS AND METHODS:

Plant Material:

The fresh leaves of Areca Catechu have been collected from the local area at Shahada and authentified by Dr. Santosh. K. Tayade, HOD of Botany, Art’s Science and Commerce College, Lonkheda, Shahada, Dist-Nandurbar (MS).

 

Worms:

Indian earthworms (Pheritima Posthuma) were used to study anthelmintic activity. The earthworms were collected from moist soil at local area at Taloda, Dist-Nandurbar. The average size of earthworm was 6 – 8 cm. Earthworm. All worms were washed with normal saline,and kept in beakers containing normal saline separately.

 

Preparation of Extracts:

Collected fresh leaves of Areca catechu were dried and crushed to coarse powder and pass it through sieve no 40 to get fine powder and subject it to maceration with Aqueous + Acetone, Alcohol and Pet. ether then dried it by using evaporator and then extract was subjected to preliminary phytochemical testing. (12, 13, 14)

 

Drugs and chemicals:

1.     Piperazine citrate.

2.     Saline solution.

 

RESULTS:

In the present study leaves of Areca catechu was evaluated for its pharmacognostic, phytochemical and pharmacological aspects which revealed the following results.

 

I] Pharmacognostic Evaluation:

1) Organoleptic Evaluation:

The results of organoleptic evaluations were given in table no.2.

 

Table no.2: Organoleptic/Macroscopic characteristics of leaves of Areca catechu

Sr. No.

Parameters

Observation of Leaf

1.

Colour

Green

2.

Odour

No odour

3.

Taste

Astringent

4.

Shape

Linear

5.

Texture

Glossy, Medium

6.

Margin

Entire

7.

Venation

Parallel

8.

Type

Even Pinnately compound

9.

Arrangement

Spiral

 


2) Microscopical Evaluation:

i) Transverse Section of Areca catechu leaves:

 

 

Fig.1. Transverse section of the leaves of Areca catechu

 


The transverse section of the leaves of Areca catechu (Fig no.1) shows the typical microscopic characteristics like upper epidermis, collenchyma, phloem, xylem, spongy parenchyma, lower epidermis etc. The characteristic are shown table no. 3.

 

Table No. 3 Microscopic characteristic of transverse section of leaves of Areca catechu

Sr. No.

Observation of T. S of leaf

1.

Upper Epidermis

2.

Collenchymas

3.

Phloem

4.

Xylem

5.

Spongy parenchyma

6.

Lower epidermis

 

(ii) Powder Analysis:

The powder of the leaves of Areca catechu shows the typical microscopic characteristics like epidermal cells, fibres, lignified vessels etc. The characteristic are shown table no. 4.

 

 

Fig. 2: Epidermal cells

 

Fig. 3: Fibres

 

 

Fig.4: Lignified vessels

 

Table no. 4: Microscopic characteristic of for leaves powder of Areca catechu

Sr. No.

Observation of areca leaf powder

1.

Epidermal cells

2.

Fibers

3.

Lignified vessels

 

 

 

 

II] Physical Evaluation:

i)     Ash values:

The total ash value, acid insoluble ash value and water soluble ash value was found to be 3.8 %w/w, 1.7 %w/w and 2.1 %w/w respectively as shown in table no.5.

 

ii)   Extractive Values:

The water soluble and Petroleum ether soluble extractive values were found to be 9.2 %w/w and 13.6 %w/w respectively as shown in table no.5.

 

iii) Loss on Drying:

The loss on drying was found to be 0.90 %w/w as shown in table no.5.

 

Table no. 5: Physical constants for leaves powder of Areca catechu

Sr. No.

Physical Constants

Results

1.

Ash Values

A)       Total Ash

B)       Acid Insoluble Ash

C)       Water Soluble Ash

 

3.8 %w/w

1.7 %w/w

2.1 %w/w

2.

Extractive Values

A)       Water soluble extractive

B)       Petroleum ether soluble extractive

 

9.2 %w/w

13.6 %w/w

3.

Loss on Drying

0.90 %w/w

 

III) Preliminary Phytochemical Screening:

The percentage yield of leaves of Areca catechu after maceration was found to be max 7.46 % (Aqueous+ Acetone extract) as compared to other extracts as shown in table no. 6.

 

Table no. 6: The percentage yield of leaves of Areca catechu after maceration

Sr. No.

Extracts

% Yield

1.

Aqueous + Acetone  extract

7.46

 

Phytoconstituents present in aqueous + acetone extract of Areca catechu leaves:

In the present investigation the extract of plant was analyzed for the presence of alkaloids, carbohydrates, glycosides, amino acids, steroids, tannins and flavonoids using standard procedures. The preliminary phytochemical investigation showed the presence of alkaloids, carbohydrates, saponins, tannins, steroids, glycosides, flavonoids as shown in table no.7.

 

Table No.7: Data showing the presence of phytoconstituents present in aqueous + acetone extract of Areca catechu leaves

Sr. No

Phytoconstituents

Aqueous + Acetone extract

1

A

B

C

D

Alkaloids

Dragendorff’s Reagent

Wagner’s Reagent

Hager’s Reagent

Mayer’s Reagent

Present

+

-

+

+

2

A

B

C

Carbohydrate

Molisch’s test

Benedict’s test

Fehling’s test

Present

+

+

+

3

A

Saponins

Foam test

Present

+

4

Tannins

Present

5

A

B

Steroids

Salkowski test

Libermann-Burchard’s test

Present

+

+

6

Glycosides

Present

7

A

B

Flavonoids

Shinoda test

Lead acetate test

Present

+

+

8

A

Amino acids

Ninhydrin’s test

Absent

-

 

IV) Pharmacological Evaluation:

1) ANTHELMINTIC ACTIVITY:

 

Table No. 8: Anthelmintic activity of Areca Catechu leaves extracts on Indian earthworms (Pheritima Posthuma)

Sr. No.

Extracts

Conc.

(in mg/ml)

Indian Earthworm

(Pheretima Posthuma)

 

 

Time of Paralysis in min (P)

Time of Death in min (D)

1

Aqueous + Acetone

25

16.85 ± 1.30

107.91 ± 3.0

50

9.95 ± 1.55

50.21 ± 2.50

75

5.64 ± 1.25

34.63 ± 1.50

100

2.29 ± 0.55

29.66 ± 1.0

2

Petroleum Ether

25

26.18 ± 1.80

137.91 ± 2.0

50

18.46 ± 1.58

99.21 ± 2.32

75

14.36 ± 1.26

64.66 ± 1.20

100

10.22 ± 0.56

49.34 ± 1.12

3

Alcohol

25

38.76 ± 1.30

158.98 ± 1.0

50

29.28 ± 1.46

120.22 ± 1.40

75

18.64 ± 1.36

84.18 ± 1.70

100

11.34 ± 0.12

69.44 ± 1.26

4

Control (Normal Saline)

-

-

-

5

Standard (Piperazine citrate)

10

9.55 ± 0.65

23.05 ± 1.35

 

 

Fig No. 5: Anthelmintic activity of Areca Catechu leaves extracts on Indian earthworm (Pheritima Posthuma)

 

AA. E = Aqueous + Acetone Extract, PE. E = Petroleum Ether Extract, Al. E = Alcohol Extract, Std = Standard, (P) = Paralysis Time, (D) = Death Time

 

RESULT:

From the above observations, higher concentration of extract produced paralytic effect much earlier and the time to death was shorter for all worms. All the extracts showed anthelmintic activity but Aqueous+Acetone extract showed better anthelmintic activity than other extracts, in dose-dependent manner giving shortest time of paralysis (P) and death (D) with 100 mg/ml concentration. Evaluation of anthelmintic activity was compared with reference standard Piperazine citrate.

 

From Table No.8, it is observed that 100mg/ml conc. of leaves of Areca catechu shown potent anthelmintic activity while 75, 50 and 25mg/ml conc. taken more time for paralysis as well as death of worms. The results are compared with the standard drug Piperazine citrate. For Aqueous+Acetone extract the time of paralysis (P) was found to be 16.85, 9.95, 5.64, and 2.29 min. at conc. of 25, 50, 75 and 100 mg/ml respectively when compared to standard i.e. Piperazine citrate (9.55 min.) at the conc. of 10 mg/ml and to normal saline as control. While the time of death (D) was found to be 107.91, 50.21, 34.63, and 29.66 min. for Aqueous+Acetone extract, at conc. of 25, 50, 75 and 100 mg/ml respectively when compared to standard i.e. Piperazine citrate (23.05 min.) at the conc. of 10 mg/ml and to normal saline as control. The results obtained indicate that Aqueous + Acetone extract of Areca catechu leaves shown significant anthelmintic activity at conc. of 100 mg/ml against Pheritima Posthuma. The results of anthelmintic activity are shown in Table No.8. The graphical representations of results were shown in Fig. No.5.

 

DISCUSSION:

Areca catechu is widely use in the traditional system of medicine for the treatment of number of diseases. The results of these investigations would helpful for proper identification, collection and investigation of the plant. The parameters determined in quantitative microscopy can be helpful to differentiated closely related species. The presence of various phytoconstituents can serve to treat diseases by using various pharmacological activities. Physical standards may be used to determine the quality of this plant in future investigation. It will also helpful to carry out further research and revalidation of its use in traditional system in medicine.

 

It also gives anthelmintic activity and the experimental evidence obtained in the laboratory model could provide a rationale for the traditional use of this plant as anthelmintic. The plant may be further explored for its phytochemical profile to recognize the active constituent accountable for anthelmintic activity.

 

CONCLUSION:

The pharmacognostic parameters, which are being reported, could be useful in the identification and standardization of a crude drug. The data produced in the present investigation is also helpful in the preparation of crude drug’s monograph and inclusion in various pharmacopoeias.

From the above results, it is concluded that the aqueous + acetone leaves extract of Areca catechu L. shows potent anthelmintic activity than standard anthelmintic drug. Further studies using in vivo models are required to carry out and establish the effectiveness and pharmacological rationale for the use of Areca catechu L. leaves as an anthelmintic drug. The drug can be further explored for the isolation and characterization of the active constituents responsible for anthelmintic activity.

 

ACKNOWLEDGEMENT:

Authors are thankful to Dr. Santosh. K. Tayade, HOD of Botany, Art’s Science and Commerce college, Shahada (M.S) for authentification of plant and to our Hon. Principal Dr. S. P. Pawar, P.S.G.V.P.M’s College of Pharmacy, Shahada (M.S) for providing all necessary facilities to carry out this research work and authors are also thankful to our librarian Mr. S. S. Patil for providing literature and necessary books, journals and magazines for completion of this research work.

 

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Received on 22.08.2019         Modified on 29.08.2019

Accepted on 03.09.2019       ©A&V Publications All right reserved

Res.  J. Pharmacology and Pharmacodynamics.2019; 11(3):95-100.

DOI: 10.5958/2321-5836.2019.00017.X