phytochemical and pharmacological investigations of ...

RESEARCH ARTICLE Department of Pharmacology

Mazumdar et.al / IJIPSR / 4 (7), 2016, 693-708 ISSN (online) 2347-2154

International Journal of Innovative Pharmaceutical Sciences and Research www.ijipsr.com PHYTOCHEMICAL AND PHARMACOLOGICAL INVESTIGATIONS OF POLYALTHIA SUBEROSA ROXB 1

Rajib Mazumdar*, 2 Md. Habibur Rahman, 2 Md. Mizanur Rahman, 2 Md. Arifur Rahman Chowdhury, 3 Md. Alamgir Hossain, 1 Md. Shamim Ahmed, 4 Poulame Das Gupta 1

Stamford University, BANGLADESH Southeast University, BANGLADESH 3 East-West University, BANGLADESH 4 University of Asia Pacific, BANGLADESH 2

Abstract The present study has established analgesic potential of P. suberosa using acetic acid-induced writhing test for visceral pain and tail immersion tests for pain mediated by central activity. The extracts significantly decreased the locomotors activity as shown by the results of the open field and hole cross tests. From the result this is observed that, bark has more CNS depressant activity than leaves parts of hydromethanolic extracts of polyalthia suberosa by using both open field & hole cross tests, which is comparable to the reference drug Diazepam at a doses of 1mg/kg. Many flavonoids were found to be ligands for the gamma amino butyric acid type A (GABAA ) receptors in the central nervous system (CNS), which led to the hypothesis that act as Benzodiazepines -like molecules. This is supported by behavioral effects in animal models of anxiety, sedation and convulsion. Preliminary phytochemical screening reveals the presence of flavonoids, so the observed CNS activity may be attributed to this compound. The extract inhibited both mechanisms of pain, suggesting that the plant extract may act as a narcotic analgesic.

Keywords : Polyalthia suberosa, Narcotic analgesic, CNS depressant activity, Flavonoids.

Corresponding Author: Rajib Mazumdar Stamford University, BANGLADESH Email: [email protected] Phone: +8801818481978 Available online : www. ijipsr.com

July Issue

693



INTRODUCTION The bark leaves and roots of some species are used in folk medicines. Pharmaceutical research has found antifungal, bacteriostatic, anti-malarial, and especially cytostatic capability of some chemical constituents of the leaves and bark. A large number of chemical compounds, including flavonoids, alkaloids and acetogenins, have been extracted from the seeds and many other parts of these plants. Flavonoids and alkaloids have shown antibacterial properties, and have been used for treatment of medical conditions, such as skin disease, intestinal worms and inflammation of the eye. Pharmaceutical products are currently in animal and human cell-line trials [1].

Acetogenins

are thought to have anti-HIV and anti-cancer effects. A wide variety of products has been developed and is available for cancer treatment. Scientific Classification Domain: Eukaryota Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Sub classis: Magnoliidae Order: Magnoliales Family: Annonaceae Genus: Polyalthia Species: Polyalthia suberosa (Roxb.) [2] Nomenclature Binomial Name: Polyalthia suberosa R. Botanical Name: Polyalthia suberosa Biological Name: Polyalthia suberosa English Name: Corky Debbar Tree Common Name: Polyalthia suberosa is locally known as:         

Humzum (Beng.), Chamkhirni (Ind.), Baling-manok (Tag.), Duhat-duhatan (Tag.), Duhat-matsing (Tag.), Duyat-nasi (Pamp.), Lanutan (Tag.), Tagputagpuan (Tag.), Munat (Ilk.).

Available online : www. ijipsr.com

July Issue

694



Plant Description Botany This plant is a shrub or small tree growing to a height of 2 to 4 meters. The leaves are narrowly obovate-oblong and 5 to 11 centimeters long. The flowers are

oblong to

solitary, pale-yellow, 1

centimeter long or less, on slender pedicels, and 1 to 2 centimeters long. The sepals and petals are slightly hairy. The fruit is numerous, ovoid or globose, 4 to 5 millimeters long, purple, fleshy and edible. Chemical Constituents and Properties The leaves contain alpha-and beta-amyrin, lupeol, beta-sitosterol, stigmasterol and ampesterol. The stems and leaves contain the triterpene, suberosol, which showed anti-HIV replication activity. The stem bark contains alkaloids, oxostephanine and nuginosine, which exhibited antibacterial activity against several Gram-positive and Gram-negative bacteria [3]. Study yielded an azaanthracene alkaloid, Kalasinamide, from the stems of P. suberosa together with the known N-trans-feruloyltyramine and N-trans- coumaroyltyramine, which show anti-tumor activity. Application Folkloric: Decoction of fresh roots used as an abortifacient (www.stuartxchange.org/Duhatmatsing.html).  Chemicals found in plant shown to be effective for the ailment medicated.  Plant itself shown to be effective for the ailment medicated [4].

MATERIALS & METHODS All the experiments of these investigations were carried out at the laboratories of the Department of Pharmacy, Stamford University Bangladesh from January, 2010 to March 2010. Preparation of Plant Sample for Experiments Collection and Identification of Cannabis indica For these present investigations, various parts of the were collected from the village Roypur of Chuadanga district. The plant leaves, bark and fruits were collected and identified by experts of Bangladesh National Herbarium Mirpur, Dhaka, where a Voucher specimen (Accession No. 32781) has been deposited for future reference. Drying of the Plant Samples The flowering tops and other adulterants were removed to get fresh sample. Then the collected samples were dried for two days in the laboratory under electric fan. Finally the leaves, bark and Available online : www. ijipsr.com

July Issue

695



fruits were dried in hot air woven at 55 0 C for 2 days and at 400 C for the next 2 days. Before drying the samples were cut and sliced where necessary. Grinding of the Dried Samples The dried samples were grounded to coarse powder with a mechanical grinder (Grinding Mill) and powdered samples were kept in clean closed glass containers pending extraction. During grinding of sample, the grinder was thoroughly cleaned to avoid contamination with any remnant of previously grounded material or other foreign matters deposited on the grinder. Extraction of the Dried Powdered Samples The dried leaves, barks and fruits of Polyalthia suberosa were coarsely powdered by a milling machine and extracted with a mixture of methanol: water (7:3, v/v) by a Soxhlet apparatus at 50 ˚C for 72 hours for each attempt of extraction of the total powder. When the powders became exhausted of its chemical constituents as evident from cycles of colourless liquid siphoning in the Soxhlet apparatus, extraction was considered to be complete. After completion of the extraction from different parts of Polyalthia suberosa, the extracts were filtered using a sterilized cotton filter. Then solvent was completely removed and obtained dried crude extract which were used for investigations.

Fig.1: Hot Extraction of Polyalthia suberosa. Phytochemical Investigation of P.suberosa Phytochemical Screening: The freshly prepared crude extract was qualitatively tested for the presence of chemical constituents. Phytochemical screening of the extract was performed using the following reagents and chemicals. First, charcoal was heated at 30o in oven for about an hour, and then it was activated. Then aquous and alcoholic extracts were prepared and decolorized them by using activated charcoal. The chemical constituents were identified by characteristic color changes using standard procedures [5]. Reagent Used for Different Chemical Tests 1. Mayer’s Reagent: 1.36 gm mercuric iodide in 60 ml of water mixed with a solution contains 5 gm of potassium iodide in 20 ml of water. Available online : www. ijipsr.com

July Issue

696



2. Dragendroff's Reagent: 1.7 gm basic bismuth nitrate and 20 gm tartaric acid were dissolved in 80 ml of water. This solution was mixed with a solution contains 16 gm potassium iodide and 40 ml water. 3. Hager’s Reagent: A 1% solution of picric acid in water. 4. Fehling's Solution A: Dissolved 34.64 gm copper sulphate in a mixture of 0.50 ml of sulfuric acid and sufficient water to produce 500 ml. 5. Fehling's Solution B: Dissolved 176 gm of sodium potassium tartarate and 77 gm of sodium hydroxide in sufficient water to produce 500 ml. Equal volume of above solution was mixed at the time of use. 6. Liberman-Burchard Reagent: 5 ml acetic anhydride was carefully mixed under cooling with 5 ml conc. Sulphuric acid; this mixture was added cautiously to 50 ml absolute ethanol with cooling. 7. Benedicts Reagent: 1.73 gm cupric sulfate, 1.73 gm sodium citrate and 10 gm anhydrous sodium carbonate were dissolved in water and the volume made up to 100 ml with water. 8. Molish Reagent: 2.5 gm of pure α -Naphthol dissolved in 25 ml of ethanol. Test for Carbohydrates 

Molisch’s test: 2ml solution of the extract of the plant material was taken in a test tube. 2 drops of freshly prepared 10% alcoholic solution of α-Naphthol was taken in test tube and thoroughly mixed. 2ml of conc. Sulphuric acid was given to flow down the side of the inclined test tube so that the acid forms a layer beneath the aqueous solution. A red or reddish violet ring was formed at the junction of the two layers if a carbohydrate was present. A dark purple solution was formed on standing or shaking.



Fehling’s test ( Standared test for Reducing sugar): 2ml of an aquous extract of the plant material was added 1ml of a mixture of equal volumes of Fehling’s solutions A & B.Boil for a few minuties.A red or brick-red precipitate confirmed the presence of reducing sugar. Table 1: Different chemical group tests of bark of Polyalthia suberosa (Hydromethanolic extract).

Sample Test for Carbohydrates: 2 drops of 10% alcoholic solution of α-Naphthol was added to 2ml of aquous extract Test for Reducing Sugar: 2ml of aquous extract

Test solution

Observation

Inference

2ml concentrated Sulphuric acid

A reddish violet ring was formed

Carbohydrate is Present

0.5mlof Feh-A + 0.5ml of Feh-B

A brick-red precipitate was formed

Reducing Sugar is present


July Issue

697


Tests for Tannins: 5ml solution of methanolic extract Test for Flavonoids: 0.5ml solution of methanolic extract Test for Saponins: 1ml solution of methanolic extract diluted with 10ml of distilled water and shake


1ml of 5% Ferric chloride solution

Greenish precipitate was formed

Tannin is present

Zinc powder + 5-10 drops of concentrated Hydrochloric acid

Red color was formed

Flavonoid is present

----------------

Lether was formed, Persists for 1-2 minuties

Saponin is present

Table 2: Different chemical group tests of bark of Polyalthia suberosa (Hydromethanolic extract) Sample Test for Steroids: 2ml of chloroform extract of the plant materials Tests for Alkaloids: 2ml solution of the extract and 0.2ml of dilute Hydrochloric acid

Test solution

Observation

Inference

1ml of concentrated Sulphuric acid

Red ring was formed

Steroid is present

1ml of Mayer’s reagent

White precipitate was formed

Alkaloid is present

2ml solution of the extract and 0.2ml of dilute Hydrochloric acid 2ml solution of the extract and 0.2ml of dilute Hydrochloric acid

1ml of Dragendroff’s reagent 1ml of Hager’s reagent

Orange-red precipitate was formed Yellow crystalline precipitate was formed

Tests for Glycosides: Small amount of methanolic extract

Few drops of aquous NaOH solution

No development of yellow color

Alkaloid is present Alkaloid is present Glycoside is absent

Tests for Glucosides: Small amount of methanolic extract

DiluteH2 SO4 +NaOH solution + Fehling’s solution 5-8drops of 10% NaOH +1-2drops of 3% copper sulphate solution.

Tests for Proteins: 1ml of hot aquous extract

No development of brick-red precipitate No development of red color

Glucoside is absent

Protein is absent

Table 3: Different chemical group tests of leaves of Polyalthia suberosa (Hydromethanolic extract) Sample Test for Carbohydrates: 2 drops of 10% alcoholic solution of α-Naphthol was added to 2ml of aquous extract Test for Reducing sugar: 2ml of aquous extract


Test solution

Observation

Inference

2ml concentrated Sulphuric acid

A reddish violet ring was formed

Carbohydrate is present

0.5mlof Feh-A + 0.5ml of Feh-B

A brick-red precipitate was formed

Reducing Sugar is present

July Issue

698



Tests for Tannins: 5ml solution of methanolic extract

1ml of 5% Ferric chloride solution

Test for Flavonoids: 0.5ml solution of methan olic extract Test for Saponins: 1ml solution of methanolic extract diluted with 10ml of distilled water and shake Tests for Proteins: 1ml of hot aquous extract

Greenish precipitate was formed

Tannin is present

Zinc powder + 5-10 drops of concentrated Hydrochloric acid + boil

Red color was formed

Flavonoid is present

----------------

Lether was formed, Persists for 1-2 min

5-8drops of 10% NaOH +1-2drops of 3% copper sulphate solution.

No development of red color

Saponin is present Protein is absent

Table 4: Different chemical group tests of Leaves of Polyalthia suberosa (Hydromethanolic extract) Sample Test for Steroids: 2ml of chloroform extract of the plant materials Tests for Alkaloids: 2ml solution of the extract and 0.2ml of dilute Hydrochloric acid 2ml solution of the extract and 0.2ml of dilute Hydrochloric acid 2ml solution of the extract and 0.2ml of dilute Hydrochloric acid Tests for Glycosides: Small amount of methanolic extract Tests for Glucosides: Small amount of methanolic extract

Test solution

Observation

Inference

1ml of concentrated Sulphuric acid

Red ring was formed

Steroid is present

1ml of Mayer’s reagent

White precipitate was formed

Alkaloid is present

1ml of Dragendroff’s reagent

Orange-red precipitate was formed Yellow crystalline precipitate was formed No development of yellow color

Alkaloid is present

1ml of Hager’s reagent Few drops of aquous NaOH solution Dilute H2 SO4 + NaOH solution + Fehling’s solution

No development of brick-red precipitate

Alkaloid is present Glycoside is absent Glucoside is absent

RESULTS & DISCUSSION RESULTS PHYTOCHEMICAL INVESTIGATION OF P.suberosa Phytochemical Screening Preliminary phytochemical screening of the crude hydromethanolic extracts of the bark & Leaves parts of Polyalthia suberosa revealed the presence of Alkaloid, Flavonoid, Saponin, Tannin, Carbohydrate (reducing sugar) & Steroid. Available online : www. ijipsr.com

July Issue

699



Table 5: Result of chemical group test of the hydromethanolic extracts of the bark & leaves parts of Polyalthia suberosa Plant Extract

Carbohydrate (Reducing Sugar)

Steroid

-

+

+

-

+

+

Alkaloid

Flavonoid

Saponin

Tannin

Glycoside

+

+

+

+

+

+

+

+

HME of P.suberosa (Bark) HME of P.suberosa (Leaves)

HME: Hydromethanolic extract; (+): Present; (-): Absent PHARMACOLOGICAL INVESTIGATION OF P.suberosa Tests for Neuropharmacological Activity Hole Cross Test The the bark & leaves parts of hydromethanolic extracts of P.suberosa extract at dose level of 100mg/kg, 200mg/kg and 400mg/kg body weight produced significant decrease of movement from its initial value during the period of hole cross experiment. The maximum decrease in movement was observed at 90 and 120 min after drug administration. Table 6: Effect of bark parts of P.suberosa extract on Hole Cross Test in mice Number of Movements Treatment

Dose (mg/kg)

0 min

30 min

60 min

90 min

120 min

Control (DMSO)

0.2ml/mouse

5.6±1.63

4.2±0.66

4.4±0.745

2±0.86

3.2±0.375

Diazepam

1mg/kg

15±1.34

7±0.835

4.6±0.925

3.6±0.87

1.4±0.745

Group-l (C.I.P.)

100mg/kg

9.2±1.325

6.4±0.58

4.6±1.015

6.2±0.975

1.8±0.485

Group-ll (C.I.P. + Sugar) Group-lll (C.I.P.+ Sugar)

100mg/kg + 300mg/kg 100mg/kg + 600mg/kg

3.8±0.835

5.2±0.675

1.6±0.77

2±1.025

1.6±0.58

4.4±1.4

2.4±1.36

2.4±0.89

2±1.173

4.2±0.4

Values are presented as mean ± SEM, where n = 5.


July Issue

700


Mazumdar et.al / IJIPSR / 4 (7), 2016, 693-708 ISSN (online) 2347-2154 Control

Diazepam

Group ll

Group lll

Group l

25

No. of holes crossed

20 15 10 5 0 0

30

60

90

120

150

Time (minutes)

Fig.2: Graphical Representation of effect of bark parts of P. suberosa extract on Hole cross test in mice. Control= 1% Tween 80 in water, 0.4 ml/mouse, Diazepam= 1mg/kg, Group l =100mg/kg, Group ll=200mg/kg, Group lll = 400mg/kg b.w. Table 7: Effect of leaves parts of P.suberosa extract on Hole Cross Test in mice Number of Movements

Treatment

Dose (mg/kg)

0 min

30 min

60 min

90 min

120 min

Control

0.4ml/mouse

17±1.968

17.8±1.917

17.6±2.280

17.2±1.557

18.4±0.909

Diazepam

1mg/kg

6±0.9354

2±0.7905

1.6±1.036

1.2±0.418

Group-l

100mg/kg

17.8±0.742

6.8±1.387

6±1.225

5.2±3.19

3.4±2.361

Group-ll Group-lll

200mg/kg 400mg/kg

8±2.85 9.8±3.13

4.4±1.151 8.2±1.084

2.8±1.636 2.25±1.652

2.6±1.204 1.2±0.418

0.6±0.446 2.8±0.894

15.4±0.090

Values are presented as mean ± SEM, where n= 5. control Group-Il

No. of holes crossed

25

Diazepam Group-lll

Group-I

20

15

10

5

0 0

30

60

90

120

150

Time (minutes)

Fig.3: Graphical Representation of effect of leaves parts of P.suberosa extract on Hole cross test in mice. Control= 1% Tween 80 in water, 0.4 ml/mouse, Diazepam= 1mg/kg, Group l =100mg/kg, Group ll=200mg/kg, Group lll = 400mg/kg b.w. Available online : www. ijipsr.com

July Issue

701



Tests for Analgesic Activity Acetic Acid-Induced Writhing Test The analgesic effect of the bark & leaves parts of hydromethanolic extracts of P.suberosa at dose level of 100mg/kg, 200mg/kg and 400mg/kg body weight on acetic acid-induced writhing in mice was exhibited. Both doses of the extract significantly inhibited writhing response induced by acetic acid in a dose dependent manner which is comparable to the reference drug Diclofenac Na. Table 8: Effect of bark parts of P.suberosa extract on Acetic Acid - Induced Writhing Test Treatment

Dose (mg/kg)

Mean

SD

SE

% of Writhing

% of Inhibition

Control Diclofenac Na Group-l Group-ll Group-lll

0.4ml/mouse 10mg/kg 100mg/kg 200mg/kg 400mg/kg

39.1 9.6 17.4 20.1 4.2

8.8769 5.1161 21.0843 16.2419 4.9573

4.4384 2.5580 10.5421 8.1209 2.4786

100 24.5524 44.5012 51.4066 10.7416

0 75.4475 55.4987 48.5933 89.2583

% of Inhibition

100

Control

Positive Control

Group 1

Group 2

Group 3

80 60 40 20 0 Group

Fig.4: Graphical Representation of effect of bark parts of P. suberosa extract on acetic acidinduced writhing test in mice. Control= 1% Tween 80 in water, 0.4 ml/mouse, Positive control= Diclofenac Na, 10mg/kg, Group l =100mg/kg, Group ll=200mg/kg, Group lll = 400mg/kg b.w. Table 9: Effect of leaves parts of P.suberosa extract on Acetic Acid-Induced Writhing Test.

4.4384 2.5580

% of Writhing 100 24.5524

% of Inhibition 0 75.4475

19.9198

9.9599

48.8491

51.1508

4.1623 16.1887

2.0811 8.0943

19.9488 64.4501

80.0511 35.5498

Treatment

Dose (mg/kg)

Mean

SD

SE

Control Diclofenac Na

0.4ml/mouse 10mg/kg

39.1 9.6

8.8769 5.1161

Group-l

100mg/kg

19.1

Group-ll Group-lll

200mg/kg 400mg/kg

7.8 25.2


July Issue

702

RESEARCH ARTICLE Department of Pharmacology Control

Mazumdar et.al / IJIPSR / 4 (7), 2016, 693-708 ISSN (online) 2347-2154 Positive Control

Group 1

Group 2

Group 3

90 80

% of Inhibition

70 60 50 40 30 20 10 0 Group

Fig.5: Graphical Representation of effect of leaves parts of P. suberosa extract on acetic acid-induced writhing test in mice. Control= 1% Tween 80 in water, 0.4 ml/mouse, Positive control= Diclofenac Na, 10mg/kg, Group l =100mg/kg, Group ll=200mg/kg, Group lll = 400mg/kg b.w. Tail Immersion Test The tail withdrawal reflex time following administration of the the bark & leaves parts of hydromethanolic extracts of P.suberosa at dose level of 100mg/kg, 200mg/kg and 400mg/kg body weight , was found to almost remain the same consistency with increasing dose of the sample which is comparable to the reference drug Nalbuphine. Table 10: Effect of bark parts of P.suberosa extract on Tail-Immersion Test Treatment Control Nalbuphine Group-l Group-ll Group-lll

Dose (mg/kg) 0.4ml/mouse 10mg/kg 100mg/kg 200mg/kg 400mg/kg

Response Times (in seconds) 0 min 2.164±0.096 2.24±0.329 2.123±0.084 2.944±0.211 2.671±0.202

30 min 2.072±0.329 5.896±0.999 2.918±0.202 3.966±0.117 3.288±0.161

60 min 2.658±0.185 12.32±1.691 4.964±0.185 5.328±0.293 5.024±0.220

90 min 2.35±0.35 12.13±1.74 6.854±0.31 6.859±0.18 6.805±0.20

Values are presented as mean ± SEM, where n= 5 Table 11: Effect of leaves parts of P.suberosa extract on Tail-Immersion Test Treatment

Dose (mg/kg)

Control Nalbuphine Group-l Group-ll Group-lll

0.4ml/mouse 10mg/kg 100mg/kg 200mg/kg 400mg/kg

Response Times (in seconds) 0 min 2.164±0.096 2.24±0.329 2.205±0.146 2.623±0.141 2.518±0.091

30 min 2.072±0.329 5.896±0.999 3.362±0.406 3.884±0.215 6.594±2.342

60 min 2.658±0.185 12.32±1.691 4.488±0.134 7.614±2.448 5.166±0.525

90 min 2.35±0.35 12.13±1.74 6.723±0.37 6.751±0.21 9.714±2.99

Values are preasented as mean ± SEM, where n= 5 Available online : www. ijipsr.com

July Issue

703



Brine Shrimp Lethality Bioassay for Cytotoxic Activity The bark & leaves parts of hydromethanolic extracts of P.suberosa were also subjected to Brine Shrimp lethality bioassay for possible cytotoxic action, which were found to be highly cytotoxic to Brine Shrimp nauplii, with LC 50 of 3.16μg/ml and 1.58 μg/ml respectively while the LC 50 of the reference anticancer drug vincristine sulphate was 0.66 μg/ml . Table 12: Brine Shrimp lethality bioassay of the bark & leaves parts of HMPS Test Solution

HMPS (Bark)

HMPS (Leaves)

Vincristine sulphate

Conc. (μg/ml)

Log Conc.

% Mortality

1.25 2.5 5 10 20

0.09691 0.39794 0.69897 1 1.3.103

40 45 50 70 75

40

1.60206

80

80 160 320

1.90309 2.20412 2.50515

85 90 100

1.25 2.5 5 10 20 40 80

0.09691 0.39794 0.69897 1 1.3.103 1.60206 1.90309

45 50 60 75 80 85 90

160 320

2.20412 2.50515

95 100

0.078125 0.15625 0.3125 0.625 1.25 2.5

-1.10721 -0.80618 -0.50515 -0.20414 0.09691 0.39794

20 30 40 50 60 80

5 10 20 40

0.69897 1 1.3.103 1.60206

90 100 100 100


July Issue

LC50 (μg/ml)

LC90 (μg/ml)

3.16

112.2

1.58

79.43

0.66

7.9

704



DISCUSSION Preliminary phytochemical screening of hydromethanolic extracts of the bark & leaves parts of P.suberosa revealed the presence of various bioactive components like, Alkaloids, Flavonoids, Steroids, Tannin, Carbohydrates (Reducing Sugar), Saponin. An important step in evaluating drug acting on CNS is to observe its effect on locomotor activity of the animal.The activity is a measure of the level of excitability of the CNS [6] and this decrease may be closely related to sedation resulting from depression of the central nervous system [7]. The extracts significantly decreased the locomotor activity as shown by the results of the open field and hole cross tests [8]. The locomotor activity lowering effect was evident at the 2 nd observation (30) and continued up to 5th observation period (120).Both hole cross and open field tests showed that the depressing acting of the extracts was evident from the 2nd observation period in the test animals at the doses of 100,200 & 400mg/kg body weight. Maximum depressant effect was observed from 3rd (60min) to 5th (120 min) observation period. From the result this is observed that, bark has more CNS depressent activity than leaves parts of hydromethanolic extracts of polyalthia suberosa by using both open field & hole cross tests, which is comparable to the reference drug Diazepam at a doses of 1mg/kg. Many flavonoids were found to be ligands for the gamma aminobutyric acid type A (GABAA) receptors in the central nervous system (CNS), which led to the hypothesis that act as Benzodiazepines -like molecules. This is supported by behavioral effects in animal models of anxiety, sedation and convulsion [9]. Preliminary phytochemical screening reveals the presence of flavonoids, so the observed CNS activity may be attributed to this compound. The present study has established analgesic potential of P. suberosa using acetic acid-induced writhing test for visceral pain and tail immersion tests for pain mediated by central activity. Acetic acid-induced writhing in mice is a model of visceral pain which is highly sensitive and useful for screening peripherally acting analgesic drugs. P. suberosa plant extracts caused dosedependent antinociception against chemical induced pain in mice. Hydromethanolic extract of the bark & leaves parts of P.suberosa were treatrd in test animals at a doses of 100, 200 & 400mg/kg body weights. The bark & leaves parts of HMPS extracts at the dose of 400 mg/kg & 200mg/kg body weight respectively was found to exhibit the highest (89.2%) & (80%) writhing response inhibitory effect respectively, where the reference drug Diclofenac Na shown about 75.5% writhing inhibitory response at a doses of 10mg/kg Acetic acid-induced writhing method is not Available online : www. ijipsr.com

July Issue

705



only simple and reliable but also affords rapid evaluation of peripheral type of analgesic action. This model represents pain sensation by triggering localized inflammatory response. Such pain stimulus leads to the release of free arachidonic acid from tissue phospholipids [10]. There are also reports on the role of flavonoids in analgesic activity primarily by targeting prostaglandin [11] and also reports on the role of tannins in antinociceptive activity [12]. As it is mentioned earlier that the phytochemical screening reveals the presence of flavonoid, tannin and reducing sugar in the plant extract. So, the observed analgesic activity may be attributed to these compounds. The Tail-immersion test are considered to be selective to examine compounds acting through opoid receptor; the extract increased mean basal latency which indicates that it may act via centrally mediated analgesic mechanism. Narcotic analgesics inhibit both peripheral and central mechanism of pain, while non steroidal anti-inflammatory drugs inhibit only peripheral pain [13]. The extract inhibited both mechanisms of pain, suggesting that the plant extract may act as a narcotic analgesic. The lethality of hydromethanolic extracts of the bark & leaves parts of P.suberosa to the Brine Shrimp nauplii were shown in. The degree of lethality shown by the extractives was found to be directly proportional to the concentration of the extractives ranging from the lowest concentration (1.25 μg/ml) to the highest concentration (320 μg/ml). This concentration dependent increment in percent mortality of Brine Shrimp nauplii produced by the P.suberosa fractions indicates the presence of cytotoxic principles in these extractives. The plant is reported to contain several phytochemical constituents most notably alkaloids (azaanthracene alkaloid) and steroids [14]. Preliminary phytochemical screening study also revealed the presence of alkaloids, and steroids. So the observed cytotoxic action may be due to the presence of such compounds. However, phenolics and flavonoids are also known to show cytotoxicity in Hoechst 33258 fluorescence assay by inhibiting cellular DNA in a concentration-dependent manner [15]. Literature review indicates that stems leaf & bark extracts of polyalthia genus show significant cytotoxicity & antitumer activity deu to abundance of alkaloid. In this study , the bark & leaves parts of HMPS extracts were found to be highly cytotoxic to Brine Shrimp nauplii, with LC 50 of 3.16μg/ml and 1.58 μg/ml respectively while the LC 50 of the reference anticancer drug vincristine sulphate was 0.66 μg/ml. . In the evaluation for general toxicity using brine shrimp, maximum mortalities took place at a concentration of 320 μg/ml whereas; least mortalities were at 1.25 μg/ml concentration. Available online : www. ijipsr.com

July Issue

706



CONCLUSION Based on the results of the present study, it can be concluded that the bark & leaves parts of crude hydromethanolic extract of Polyalthia suberosa Roxb possesses remarkable cytotoxic, CNS depressant and analgesic potential.Various phytochemical constituents like alkaloid, flavonoid, tannin, saponin, carbohydrate (reducing sugar) and steroid present in the plant, as evident from phytochemical analyses, may be responsible for the observed bioactivities. Literature review of Polyalthia suberosa indicated that phytochemical and pharmacological investigations have been done on this plant extract previously. In this study, effective cytotoxic activity was found, which are similar to the result of the previous investigations done by the others. In addition, positive result in cytotoxic activity test led us to the inference that the Plant extract may contain bioactive compounds which may aid ongoing anticancer drug discovery from floristic resources. However, all the investigations were preliminary type and more sophisticated technology should be adopted. More precise methods should be adopted for pain sensation test. During CNS depressant experiment & analgesic activity test surrounding environment was maintained properly. Since the extract is reported to contain a myriad array of compounds, it is difficult to ascribe these observed activities to any specific group of compounds. Hence, further studies are suggested to be undertaken to pinpoint the exact compound(s) and to better understand the mechanism of such actions scientifically.

REFERENCE 1. Mazumder MEH, Alam B, Alam MA, Jamila M and Muhsin MDA. 2009. Investigations of secondary metabolites, antioxidant and cytotoxic activities of the leaf extract of Portulaca oleracea L. Bangladesh J Life Sci. 21(1): 17-22.

2. Takagi K, Watanabe M and Saito H. 1971. Studies on the spontaneous movement of animals by the hole cross test: Effect of 2-dimethylaminoethane. Its acylates on the central nervous system. Jpn J Pharmacol. 21: 797.

3. Lang S and Groth U.2008. Total synthesis of Azaanthracenones Natural Products Kalasinamide, Geovanine, Marcanine A. University of Konstanz Germany, Fachbereich Chemie and Konstanz Research School Chemical Biology.

4. Tuchinda P, Pohmakotr M, Reutrakul V, Thanyachareon W, Sophasan S, Yoosook C, Santisuk T and

Pezzuto MJ.2001.2-substituted furans from Polyalthia suberosa. Planta

Med.67: 572-575.


July Issue

707



5. Toma W, Graciosa JS, Hiruma-Lima CA, Andrade FDP, Vilegas W and Souza Brita ARM. 2003. Evaluation of the analgesic and antiedematogenic activities of Quassia amara barks extract. J Ethnopharmacol. 85: 19-23.

6. http://www.icuc-iwmi.org/files/News/Resources/Factsheets/annona.pdf 7. Pal S, Sen T and Chaudhuri AK. 1999. Neuro-psychopharmacological profile of the methanolic fraction of Bryophyllum pinnatum leaf extract. J. Pharm. Pharmacol. 51:313-18.

8. http://commons.wikimedia.org/wiki/Category:Polyalthia_ suberosa 9. http://en.wikipedia.org/wiki/Annonaceae 10. Dan MS, Dan SS, Mukhopadhyay P and Mukherjee MK.1985. Chemical Investigation of Some Annonaceae Species. Pharmaceutical Biology.23:73-76.

11. www.crac-grn.com/files/Char_Annonaceae.pdf 12. www.bpi.da.gov.ph/Publications/mp/pdf/d/duhat-matsing.pdf 13. Thomas KS, Nicoll JP and Coleman P. 2001. Use and expenditure on complementary medicine in England: a population based survey. Compl Ther Med. 9: 2-11. Chopra RN, Nayar SL and Chopra IC. 1956. Glossary of Indian medicinal plants Council Sci. Industrial Res. New Delhi. 1: 197.

14. www.stuartxchange.org/Duhat-matsing.html 15. Federsel HJ. 2003. Logistics of process R&D: Transforming laboratory methods to manufacturing scale. Nat Rev Drug Discov. 2: 654-664.


July Issue

708