Evaluation of Anti Bacterial and Anti Platelet Activity of Ethanolic Leaf Extract of Physalis angulata

Jafferi SAH¹, ^*Sandhya S¹, Vinod KR¹, Narender Prasad D¹ and Venkataramana K²

¹Nalanda College of Pharmacy, Nalgonda, A.P

²ASN Pharmacy College, Tenali, A.P

ABSTRACT:

There has always been a closed association between plants and human beings through ancient time and till date. Physalis angulata is an annual herb indigenous to many parts of the tropics, including the Amazon. It can be found on most continents in the tropics, including Africa, Asia, and the Americas. It grows up to 1 m high, bears small, cream-colored flowers, and produces small, light yellowish-orange, edible fruit sometimes referred to as Cutleaf Groundcherry. It is a popular medicine used due to its anti-inflammatory effects, anti tumor effects, immunomodulatory effects etc. The present study is to evaluate the anti bacterial and anti platelet activity of the ethanolic leaf extract of Physalis angulata. From the phytochemical screening it was found that the plant is mainly constituted with phenols, steroids and flavonoids .The anti bacterial activity was performed by well diffusion method on various concentrations of the extract. Gentamycin was used as the standard and ethanol as the control. In the antiplatelet activity study, the platelet was induced using adenosine di phosphate. Nephlometer was used to observe the aggregation. It was observed that the ethanolic leaf extract showed pronounced anti platelet activity. But the anti bacterial activity was found to be false positive.

KEYWORDS: Physalis angulata, anti-platelet, anti bacterial, nephlometer

INTRODUCTION:

Plants have been associated with the health of mankind from time immemorial .In the past sickness was viewed as a punishment from the god’s and hence was treated with prayers and rituals that included what may have been considered “magic portion” prepared from local herbs. Ethno-botany got its prominence as a science of relationship between primitive society and their environment. India has been described as the “Melting Point” races and tribe’s¹. It has a treasure of ethno-botanical knowledge due to presence of multi ethnic groups of ancient lineage and diverse vegetation. About 7.5% of India’s population follows a distinct life style identified as tribal culture. Tribal people of Andhra Pradesh are endowed with a deep knowledge concerning the use of wild plants as sources of food and medicine Tribe’s constitute an average of 6% of total population of Andhra Pradesh. Andhra Pradesh is the home land of 33 tribes generally distributed in hilly and interior forest areas .Major primitive tribes are Chenchu, Kond, Kutia Khond, Kolam .The Sugalis and Lambadies are the largest tribes found in Andhra Pradesh². Most of the above mentioned tribes of Andhra and many other Indian tribes use Physalis angulata for its medicinal properties. Ayurveda and Unani systems also have a very especial reference mentioned in their literature.

P. angulata is an annual herb indigenous to many parts of the tropics, including the Amazon. It can be found on most continents in the tropics, including Africa, Asia, and the Americas. It grows up to 1 m high, bears small, cream-colored flowers, and produces small, light yellowish-orange, edible fruit sometimes referred to as Cutleaf Groundcherry. Fruit is about the size of a cherry tomato, and like tomatoes, it contains many tiny edible

seeds inside P.angulata propagate easily from the many seeds the fruit contains; spontaneous clumps of plants can be found along river banks and just about anywhere the soil is disturbed and the canopy is broken³.

The plant is found to have promising anti inflammatory effect, immunomodulatoty effect, anti tumor activity, anti cariogenic, anti oxidant activity anti noniceptive, trypanosidal activity^4-11. P.angulata mainly consists of secosteroids, phenolic compounds and flavonoids. Some of the constituents established are Physalins B, D, F and G, withanolides and seco-steroids^12-15.

Blood platelets are involved in haemostasis. The normal haemostatic system limits blood loss by precisely regulated interactions between components of vessel wall, circulating blood platelets and plasma proteins. Platelets can adhere to the walls of the blood vessels, release bio reactive compounds and aggregate to each other. These properties increase to a well established level in conditions of arterial thrombosis and atherogenesis. Several agonists such as ADP, thrombin, collagen and serotonin induce the release of arachidonic acid, after phospholipase activation through calcium mobilization.Several drugs have been developed to block the different steps in platelet activation pathways. Inhibition of platelet function by Aspirin has been very well established.The present study is to give a highlight on the anti bacterial and anti platelet activity of the ethanolic leaf extract of the plant.

MATERIALS AND METHODS:

Physalis angulata was collected in the month of November and December from the surrounding areas of Nalgonda and Ranga Reddy district, A.P, India. The plant species was shade dried and stored. It was identified and authenticated by Department of Botany, Osmania University, Hyd. Preparation of Herbarium was submitted and the plants were certified as Physalis angulata L. ; Family – Solanaceae; Voucher no : 00490 (OUAH)

Chemical requirement:

Distilled water, ethanol, n-butanol, n-hexane, ethylacetate, n-hexane, toluene, Hydrochloric Acid, sulphuric acid, sodium hydroxide trisodium citrate, (SD Fine chemicals,India) ,ADP Adenine-di-phosphate(Sigma aldrich, USA) ,sterile saline(Baxter ,India),Nutrient agar(Mc kine,India).Other reagents used were of laboratory grade and obtained from various other commercial sources.

Equipment requirement:

Soxhlet apparatus, Rotary Vacuum evaporator (Indosati,India), Heating mantel (Bio-technics,India), Nephlometer (Indosati,India), Micro pipettes ,Autoclave, Incubator Oven, Laminar air flow, (SVI,India) ,Refrigerator(Whirlpool,India) Centrifugator, Silica crucible, microscope.

Preparation of the extract:

200gm of P.angualta was taken and extracted with ethanol for 18 hrs .The thick mass obtained was evaporated with help of vacuum rotary evaporator and % yield was calculated. The percentage yield of P.angulata obtained was found to be 14.59%w/w.

Preliminary chemical screening: ^{16, 17}

The extract was subjected to qualitative chemical test for the identification of various plant constituents. The extract showed presence of steroids, flavonoids , tannins and phenols.

Antibacterial activity:

Micro organisms: Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. These organisms were procured from the Dept. Of Microbiology, ANGARU, Hyderabad.

All petri dishes (diameter 86 mm) and graduated measuring pipettes were dry heat sterilized in a Autoclave at 120 ⁰C for 1.5 hrs. Media were steam sterilized at 121⁰C(15 psi) for twenty minutes in an autoclave. All plates were prepared with an equal thickness of nutrient agar. Test organisms were grown overnight at 37⁰ C in Nutrient agar broth.Well plate technique was used to perform the antibacterial assay. Four test concentrations of the plant was made of 250, 500, 750 and 1000µg/ml along with a standard Gentamycin and ethanol as the control. After overnight incubation at 37⁰C in the incubator, the zones of inhibition were observed.

Antiplatelet activity:¹⁸

Preparation of different concentration of the extract: The crude extract of the plant was taken and a stock solution was prepared from which three concentration of 50, 200 and 400 mcg/ml were obtained.

Isolation of platelets Nine parts of blood collected by venipuncture were drawn into one part of 3.8% trisodium citrate. Platelet-rich plasma (PRP) was prepared by centrifugation at 400 × g, for 6 minutes, at 22° C. Platelets were adjusted to 3.0 × 10 ⁸ cells/mL with sterile saline.

Measurement of platelet aggregation The platelet aggregation was determined by the turbidimetric method using a Nephlometer. Aliquots of 400µL of a human platelet suspension were transferred into a small cuvette and stirred at a constant speed of 180 × g at 37° C. The platelets were pre-incubated with the ethanolic extract of different fractions of P.angulata and sterile saline (vehicle) for 5 minutes at 37° C, before the addition of adenosine diphosphate (ADP-6µM). The extent of aggregation (%) was recorded continuously for 5 minutes after the addition of the agonist.

Data analysis:Results were expressed as mean ± standard error of the mean (S.E.M.). Statistical evaluation by Student's t-test was performed. The computer software used was Graph Pad Prism 5.0.

RESULTS AND DISCUSSION:

Anti bacterial activity: Ethanolic extract of P.angulata showed no antibacterial activity towards the bacterial strains of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus.

P.angulata has failed to show any anti bacterial activity against all the four bacterial strains which were assayed . This result was further substantiated by Silva, et.al (Mem,Inst.Ostwaldo Cruz ) in 2005 of S. American conducted a study on complex physalin metabolites present in the capsules of the fruit of Physalis angulata L. have been isolated and submitted to a series of assays of antimicrobial activity. P.angulata showed optimal antimicrobial activity against S.aureas, but was inactive against other bacterial strains¹⁷.

Fig no: 1 Image showing antibacterial assay for P.anagulata

Table no: 1 Observation for antibacterial activity of P.angulata.

Type of Bacterial strain	250mcg/ml	500mcg/ml	750mcg/ml	1000mcg/ml	Control	Gentamycin (50mcg/ml) MIC in mm
Escherichia coli	-	-	-	-	-	6
Bacillus subtilis	-	-	-	-	-	6.5
Pseudomonas aeruginosa	-	-	-	-	-	6.5
Staphylococcus aureus	-	-	-	-	-	6

Table no: 2 Antiplatelet activity of P.angulata

	CONTROL	400 Mcg/ML	200 Mcg/ML	50 Mcg/ML
MEAN	1000*	177.*	215*	285*
SEM	0	1.24	4.18	3.74
N	3	3	3	3

Antiplatelet activity:

Regarding platelet aggregation induced by ADP, it was found that the ethanolic extract of P.angulata was effective in inhibiting the platelet aggregation stimulated by ADP (6 µM). This aspect establishes the claim of the anti inflammatory activity of the plant.

The values represent the percentage inhibition of ethanolic extract obtained from P.angulata leaf. Control of platelet aggregation induced by ADP(6Mcg/ml),*Represents value in terms of n.t.u(Nephlo-turbidometric unit)

Student's t-Test:

t = 0.567 ; sdev= 130.

degrees of freedom = 4 The probability of this result, assuming the null hypothesis, is 0.96

Group A: Number of items= 3

50.0 200. 400.; Mean = 217.

95% confidence interval for Mean: 9.070 thru 424.3

Standard Deviation = 176.; Hi = 400. Low = 50.0

Median = 200.

Average Absolute Deviation from Median = 117.

Group B: Number of items= 3

177. 215. 285.; Mean = 223.

95% confidence interval for Mean: 15.07 thru 430.3

Standard Deviation = 52.2; Hi = 285. Low = 177.

Median = 210.

Average Absolute Deviation from Median = 34.0

ACKNOWLEDGEMENT:

The authors humbly acknowledge the principal and the management of Nalanda College of Pharmacy for the support throughout this work.They also express sincere thanks to the Department of Microbiology, ANGARU, Hyderabad, for providing the bacterial strains.

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Received on 01.12.2009

Accepted on 10.01.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(1): Jan.-Feb. 2010, 67-69