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Averrhoa carambola, L.

leaf extract

INTRODUCTION

Medicinal plants, the World’s oldest known healthcare products,

play a key role in traditional medicines. They are not only used for

primary health care, many widely used pharmaceuticals are derived from

plants and other natural resources (G.R.K.Sharma, 2006).

The history of medicine dates back perhaps to the origin of human

race. Rig veda written between 4500-1600 BC is the oldest repository of

human knowledge in India. Later, Suhsruta Samhita and Charaka Samhita

dealt with various drugs (Jain,S.K., 1987). The Sushruta Samhita contains

184 chapters and description of 1120 illnesses, 700 medicinal plants, a

detailed study on Anatomy, 64 preparations from mineral sources and 57

preparations based on animal sources (Raju V.K., 2003). The Charaka

Samhita was recorded several thousand years ago from the

Teachings of the sage Punarvasu Atreya. The Charaka Samhita is

believed to have arisen around 400-200 BC. Charaka contains over 8,400

metrical verses, which are often committed to memory, in toto, by

modern medical students of Ayurveda. It presents most of the theoretical

edifice of Ayurveda and concentrates on the branch of Ayurveda called

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Averrhoa carambola, L. leaf extract
kayachikitsa (internal medicine) (Menon, I A., et al., 1969).

Traditional healthcare systems using medicinal plants can be

recognized and used as a starting point for the development of novelties

in drugs. The volatile components of medicinal plants and herbs are

extracted that can be used for various purposes.

WHO had listed over 21,000 plant species used around the World

for medicinal purposes (NBPGR Booklet, 2006). Overall 119 plant

derived prescription drugs are commonly used in different countries, 74%

of which were discovered due to chemical isolation of active compounds

of plants used in traditional system of medicine (Sharma,G.R.K., 2006).

India is ranked no.8 in World Biodiversity. About 70% of India’s

medicinal plants are found in Tropical area and the rest in temperate and

alpine areas. Macrostudies showed that a large percentage of known

medicinal plants occur in the dry and moist deciduous vegetations as

compared to the evergreen or temperate habitats (Tiwari, 2000).

In some countries like India, pharmaceutical companies are already

marketing preparations of tablets, capsules, etc. they are made directly

from appropriate plant extracts for the treatment of specific diseases like

Hepatitis. Medicinal plants and herbs contain substances known to

modern and ancient civilizations for their healing properties. Until

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Averrhoa carambola, L. leaf extract
development of chemistry, particularly, of the synthesis of organic

compounds in the nineteenth century, medicinal plants and herbs were

sole source of active principles capable of curing man’s aliments. They

continue to be important to people that do not have access to modern

medicines and moreover, modern pharmaceuticals rely heavily on the

same active principles (Yusuf et al., 1999).

Drug discovery, ethnobotany and traditional and indigenous

medicines have long been basic to medicinal plant research. As new uses

for medicinal plants have been discovered and popularized, sustainability

has become increasingly an issue; concern over the growth in biopyracy

goes hand in hand with the critical need for conservation of both species

and habitat (Lewis, et al., 2003).

The present study is concentrated on Averrhoa carambola, L. of

the family Geraniaceae. This plant is most common in African countries

and its fruits are used as a vegetable and in desserts. In India, even though

it is a native species, is less common among people as a food source. But

the analysis of star fruits revealed the presence of high amount of Dietary

fibers, protein, Vitamin C, Potassium, etc.(Facciola, et al., 1990). The

phytochemical screening as well as antimicrobial and anthelmintic assays

was carried on the leaves to reveal its potential as a good source of drug.

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Averrhoa carambola, L. leaf extract
Plant Description

1.1. Taxonomy

Current name: Averrhoa carambola (The generic name is after

Averrhoes (1126-98), the widely known Arab Philosopher. The specific

name, ‘carambola’, is said to have come from Malabar and was adopted

early by the Portuguese).

Authority : L.

According to Bentham and Hooker’s classification

Kingdom : Plantae

Class : Dicotyledonae

Sub-class : Polypetalae

Series : Disciflorae

Cohort : Geraniales

Family : Geraniaceae

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Averrhoa carambola, L. leaf extract

K5C5A(5+50) G(5)

Synonym(s)

Averrhoa acutangula., Stokes

Sanskrit Synonyms

Karmaranga, Karuka

1.2. Common names

Malayalam : Chaturappuli, Irumbanpuli, Karappola

Hindi : kamrakh, kamranga

English : carambola, star fruit, Carambola apple, Karazola,

Coromandal gooseberry

Filipino : balimbing

French : blinblin longue, carambolier, cornichon du pays

Indonesian : belimbing manis

Malay : belimbing manis

Mandarin : yongt’o

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Averrhoa carambola, L. leaf extract
Spanish : carambola, carambold, carambole, jalea

Thai : ma fueang

Vietnamese : khê

1.3. Morphology

Tree to 30 ft (Fig.1); young parts finely pubescent; leaf 5-11,

ovate-elliptic, short pointed, mostly 1-2 in. long, nearly glabrous beneath;

flowers in the leaf axils(Fig.2); petals about ¼ in. long, white-marked

purple; 5 stamens without anthers; fruit 2-5 in. long, ovoid or ellipsoid, 3-

5 deep ribs, yellow or yellow brown, very pulpy, mildly acidic or even

sweet when ripe, fragrant (Fig.3&4)(Bailey,L.H., 1948)

1.4. HABITAT

Grows best in the hot, humid tropics but will tolerate some cool

weather. Young plants may be killed or badly damaged by frost, while

mature specimens can withstand temperatures as low as -30 C for short

periods, with some damage to branches and leaves. They can also tolerate

dry periods and some wind if it is not too cold.

1.5. Geographic distribution

Native : Indonesia, Malaysia, India, Sreelanka

Exotic : Australia, Brazil, Cambodia, China, Colombia, Dominican

Republic, Israel, Japan, Laos, Myanmar, Philippines,

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Averrhoa carambola, L. leaf extract
Singapore, Taiwan, Province of China, Thailand, Trinidad

and Tobago, Uganda, United States of America, Vietnam

1.6. Biophysical limits

Altitude : 0-900 m

Soil type : Prefers deep, well-drained clay loams but can grow

successfully on sandy soils and heavy clays.

Ayurvedic Medicinal Properties

Rasa : Amla

Guna : Guru, Snigda

Virya : Ushna

1.7. Medicinal Properties of the Plant as per Ayurveda

Plant pacifies vitiated kapha, pitta, skin diseases, poison, pruritus,

worm infestations, diarrhea, vomiting, hemorrhoids, intermittent fever,

over perspiration and general debility.

In India, the ripe fruit is administered to halt hemorrhages and to

relieve bleeding hemorrhoids. The dried fruit or the juice may be taken to

counteract fevers.

Other medicinal uses

A conserve of the fruit is said to allay biliousness and diarrhea and

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Averrhoa carambola, L. leaf extract
to relieve a"hangover" from excessive indulgence in alcohol. A salve

made of the fruit is employed to relieve eye afflictions. In Brazil, the

carambola is recommended as a diuretic in kidney and bladder

complaints, and is believed to have a beneficial effect in the treatment of

eczema. In Chinese Materia Medica it is stated: "Its action is to quench

thirst, to increase the salivary secretion, and hence to allay fever." A

decoction of combined fruit and leaves is drunk to overcome vomiting.

Leaves are bound on the temples to soothe headache. Crushed leaves and

shoots are poulticed on the eruptions of chicken-pox, also on ringworm.

The flowers are given as a vermifuge. In southeast Asia, the

flowers are rubbed on the dermatitis caused by lacquer derived from Rhus

verniciflua, Stokes. Burkill says that a preparation of the inner bark, with

sandalwood and Alyxia sp., is applied on prickly heat. The roots, with

sugar, are considered an antidote for poison. Hydrocyanic acid has been

detected in the leaves, stems and roots. The powdered seeds serve as a

sedative in cases of asthma and colic. (Morton,J., 1987).

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Averrhoa carambola, L. leaf extract
REVIEW OF LITERATURE

Phytochemical properties

Β- Anisaldehyde and β- sitosterol were isolated and elucidated

from carbontetrachloride and chloroform soluble portions of methanolic

extract of stem bark of Averrhoa carambola (Md. Masum Mia et al.,

2007).

On GC/MS analysis of chloroform soluble fraction of leaves, high

percentage of esters (45.06%) dominated by byranillin acetate (7.63%) in

addition to pentacosane(11.12%) were revealed. Similarly GLC of

saponifiable and unsaponifiable fractions of petroleum ether extracts of

both stem and leaves show that high percentage of unsaturated fatty acids

(31.87%) is present in leaves than in stem and TSFA in stem is more than

those present in leaves. Β- amyrin, stigmasterol and β- sitosterol

represented major constituents of unsaponifiable fraction (Tadros, S.H et

al., 2004)

The Total Phenolic Content (TPC) analysis of fruit of Averrhoa

carambola (star fruit) revealed that the acetone extracts contain highest

amount of total phenolics followed by ethanolic and methanolic extracts

(C.F. Yap et al., 2009)

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Averrhoa carambola, L. leaf extract
Volatile constituents present in methaylchloride extract of star

fruits showed the presence of methyl anthranilate (cause aroma), ethyl

sorbate, ethyl butyrate, 6-methyl-5-hepten-2-one, , 6-methyl-5-hepten-2-

ol, phenyl ethylacetate, limonene, phenylethyl alcohol,

etc.(Wilson,C.W.,et al.,1985)

Polyphenyloxidase was isolated from starfruit causing browning of

fruits(Tengku Adnan,T.A.B., 1986). Three insoluble Fibre-Rich Fractions

(FRFs) including insoluble dietary fiber, alcohol-insoluble solid and

water-insoluble solid isolated from star fruits could retard glucose

diffusion, postpone release of glucose from starch and inhibit the activity

of α –amylose to different extent (Chi-Fai Chau et al., 2003).

Alkyl phenols like 2,5-dimethoxy-3-undecylphenol and 5-

methoxy-3- undecylphenol and benzoquinones like 5-o-methylembelin

and 2-dehydroxy-5-o-methylembelin were isolated and elucidated from

the wood of Averrhoa carambola.

Another member of the family Oxalidaceae, Oxalis erythrorhiza

contain 3-heptadecyl-5-methoxy phenol and embelin in its

leaves.(Gabriela Egly Feresin, et al., 2003). Similarly purple leaves of

Oxalis triangularis contain anthocyanins like Mavidin-3-o(4-o-malonyl-

α-rhamnopyranosyl)-β-glucopyranoside)-5-o-β-glucopyranoside(Torgils

Fossen, et al.,2005).

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Averrhoa carambola, L. leaf extract
Three c-glycosylflavanones (isovitexin, isoorientin and swertisin)

were present in the leaf extract of Oxalis corniculata(Hiroki mizokami, et

al., 2007).

Antimicrobial properties

Carbon tetrachloride soluble fraction and petroleum ether soluble

fraction of methanolic extract of bark of Averrhoa carambola showed

moderate inhibitory growth while chloroform solvent showed inhibitory

zone in growth of E.coli. Similarly Vibrio mimicus show moderate

inhibitory growth in petroleum ether extract and very low inhibitory

growth in chloroform and carbon tetrachloride solvents (Md.Masum

Mia,et al., 2007).

Klebsiella sp. and Staphylococcus aureus possess inhibitory zones

of growth in aqueous extract of stem of Averrhoa carambola

(B.Sripanidkulchai,et al., 2002).

Ethanolic extract of leaf of Averrhoa bilimbi showed Minimum

Inhibitory Concentration (MIC) against Bacillus egaterium and E.coli

(Mackeen,M.M, et al., 1997).

Similarly methanolic extract of leaf of Averrhoa bilimbi showed

less potent antimicrobial activity against Salmonella paratyphi and

Bacillus megaterium than fruit extract. Same is in the case of

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Averrhoa carambola, L. leaf extract
Staphyllococcus aureus, Bacillus subtilis and Vibrio parahemolyticus

(Sreedam Chandra Das, et al., 2011).

Antioxidant properties

Methanolic extracts of star fruit exhibited high percentage

inhibition of DPPH radicals, Ferric Reducing Antioxidant Property(FRAP

value), antioxidant capacity and total Phenolics compared to aqueous

extracts and control after examining the effect of sonication treatment

(Annegowda,H.V., et al., 2007).

Ethanolic extract of star fruit showed a moderate potency in

reducing lipid peroxidation (37.08%) and increased the antioxidants-

TAA, SOD CAT, GsH and GPX ( Rupal, A.V., et al., 2011).

Anthelmintic properties

Averrhoa carambola showed anthelmintic activity in dose

dependent manner giving shortest time of paralysis and death with 100

mg/ml concentration (Anisha.N.Shah, et al., 2011).

Pharmacologic and medical studies

Averrhoa carambola tea extract show a very alternation of cell

cycle of Allium cepa,L., but neither induced an increase in number of

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Averrhoa carambola, L. leaf extract
chromosomal damage in bone marrow cells of Wistar rats nor alter their

cell cycle (Pimenta Vincentini V.E., et al., 2001).

Aqueous leaf extact of Averrhoa carambola depresses Guinea pig

atrial inotropism due to reduction of L-type calcium current

(Vasconcelos,C.M.L., et al., 2008).

The leaves from carambola have been utilized for the production of

herbal medicine Glico-Vitae (GV) indicated for non-insulin dependent

Diabetes mellitus (NIDDM)(Marilene Provasi, et al., 2001).

Star fruit intoxication can occur in patents with moderate Chronic

Renal Impairment (CRI) resulting in hiccups, mental confusions, coma,

etc. (Alexandre Herbland, 2007)

Neurotoxic fraction obtained from star fruit specifically inhibited

GABA binding (Carolino,R.O.G, et al., 2005).

Oxalate may play a key role in star fruit neurotoxicity in

nephrectomized rat and prpbably in uremic patients (Hua-Chang Fang, et

al., 2007)

Aqueous extract of Averrhoa bilimbi controlled rise in glucose in

blood of mice that has induced Diabetes (Giuseppina Negri, 2005). It also

prevents platelet aggregation and thrombosis in blood vessels of

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Averrhoa carambola, L. leaf extract
streptozotocin- induced diabetic rats (Pushparaj, et al., 2001).

Amentoflavone, a biflavanoid from Biophytum sensitivum could

inhibit the process of angiogenesis (Guruvayoorappan,C, et al., 2009).

Decoction of whole plant of Biophytum pertersianum has a

significant antagonistic effect on caffeine- induced calcium release from

sarcoplasmic reticulatum and hence can be used as antihypertensive

(Mouzou,A.P, et al., 2010).

Cytotoxicity

Oral administration of Averrhoa carambola juice induced serum

ALT (ALanine aminoTransferase) level an increase of 38% for rats

(ZyKhoo, CC Teh, et al., 2010).

Alcoholic extracts of stems of Averrhoa carambola exhibited

selective activity against U251 (brain tumour cell line) and that of leaves

was effective against Hepg2 (liver carcinoma cell line) only (Tadros,S.H

and Sleem,A.A., 2004).

Economic importance

 Used for making fresh juice, wines, etc.

 Ingredient in Antioxidant Green Tea as a Free Radical Fighter.

 Juice as a Beverage with high phytochemicals / phytonutrient

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Averrhoa carambola, L. leaf extract
values: Vitamin C, Antioxidants, flavanoids, phytoferols etc.

 As a cosmetic ingredient especially in moisturicing and anti-ageing

creams

 Used to make beauty soaps in Thailand.

 Cleaning: The acid type carambola dissolves tarnish and rust,

occasionally used for cleaning and polishing metal.

 Stain remover:, fruit juice is used in washing clothes and to remove

spots and stains. Contains potassium oxalate which is used for dyeing

(stuartxchange.org).

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Averrhoa carambola, L. leaf extract
MATERIALS AND METHODS

3.1. Plant material

The material was collected from the compounds of Napier

Museum, Thiruvananthapuram. It was identified using Flora of the

Presidency of Madras (J.S.Gamble, 1915). The plant material was

authenticated at the Herbarium of Botany Department, University

College, Thiruvananthapuram were voucher specimen (no. 2316) was

deposited.

3.2. Preparation of extract

The phytochemicals present in the plant material was extracted by

distillation method using soxhlet apparatus with methanol as solvent.

About 700 g of plant material i.e., leaves were collected and washed well

to remove dirts. Moisture was removed by blotting with a cotton cloth

and shade dried for 10 days. The dried leaves were powdered and 50 g of

powder sample was packed in a thimble and kept in soxhlet apparatus.

About 300 mL of methanol was poured into the bottom flask of soxhlet

apparatus. The whole apparatus was kept over a heating mantle and was

continuously heated for 8 hours at 650 C (Boiling point of methanol). The

extract was concentrated to dryness and the residues were transferred to a

pre-weighed sample bottle and were stored in a desiccator for further

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Averrhoa carambola, L. leaf extract
studies (Harborne, 1973).

3.3. PHYTOCHEMICAL SCREENING OF PLANT EXTRACT

Different biochemical parameters like reducing sugar, flavonoids,

steroids and terpenoids, tannins, caumarins, saponins and glycosides were

analysed.

3.3.1. Qualitative Analysis

Test for reducing sugars (Fehling’s test)

The aqueous methanolic extract was added to boiling Fehling’s

solution in a test tube. A brick red colour indicates the presence of

reducing sugars.

Test for flavonoids (Shinoda test)

Take 1 mL of extract and add a few magnesium turnings, followed

by addition of conc. HCl drop by drop. Development of pink colour

indicates the presence of flavonoids.

Test for saponins

5 mL of the extract was shaken with 5 mL of distilled water and

heated to the boiling point. Froathing indicates the presence of saponins.

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Averrhoa carambola, L. leaf extract
Test for alkaloids

To 5 mL of warmed extract add 10 mL 2% HCl, warmed and

filtered. 1 mL of aliquot was treated with a few drops of Dragendroff’s

reagent. Orange brown precipitate indicates the presence of alkaloids.

Test for steroids and terpinoids

In 2ml of dry and chloroform dissolved extract, add a few drops of

acetic anhydride and conc.H2SO4 and keep undisturbed for few minutes.

Formation of reddish brown colour indicates the presence of steroids

while pink colour indicates the presence of terpinoids.

Test for Tanins

To 1 mL of the extract, add 2 drops of 5% FeCl3. Presence of dirty

green indicates the presence of tannins.

Test for coumarins

1 mL of the extract was dissolved in methanol followed by the

addition of a few drops of alcoholic NaOH. Conc.HCl was added through

the sides of the test tubes which shows the appearance and disappearance

of yellow colour, indicating the presence of coumarins.

Test for Glycosides (Killer-Killani test)

To 0.5 g of the extract diluted to 5 mL with distilled water, add 2

mL of glacial acetic acid followed by a drop of Ferric chloride solution.

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Averrhoa carambola, L. leaf extract
This was underplayed with 1 mL of H2SO4. A brown ring at the interface

indicates the presence of glycosides.

3.3.2. Identification of some compounds by HPLC (High

Performance Liquid Chromatography)

Apparatus and Chromatographic conditions

A supercritical fluid extractor SFE-2 (Applied Separation, USA)

which is capable of pressure up to 680 bar and temperature up to 240ºC,

static and dynamic extraction with flow from 0 to 10 L/min (gaseous

carbon dioxide) and extraction vessels from 5 ml to 1l were used. An

Agilent 1200 liquid chromatograph system (Agilent technologies, CA,

USA) consisting of binary pump, an auto-sampler and diode-array

detector was used.

The column configuration consisted of an Agilent Zorbax Extend

reversed-phase C18 column (250 mm × 4.6 mm, 5 μm). Detection

wavelength was set at 220 nm. The mobile phase consisted of A

(methanol) and B (deionized water), using a linear gradient: 0-40 min

(85% A), 40-60 min (85% A-95% A). The flow rate was 1.0 ml/min. The

column temperature was maintained at 30 ºC.

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Averrhoa carambola, L. leaf extract
Preparation of standard solutions - Averrhova

A mixed standard stock solution containing

a) Epigallocatechin

b) Catechin

c) Caumarin

d) Squamostatin

e) Squamocin

f) Ascorbic acid

g) Gallic acid

h) Gallotannin

i) Abscisic acid

j) Vomifoliol

k) Proanthocyanidin

l) Tocopherol (m)nerol

m) Linalool

n) Geraniol

o) Terpenol

p) Roseoside

q) Methyl benzoate

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Averrhoa carambola, L. leaf extract
Was prepared in methanol. Working standard solutions were

prepared by diluting the mixed standard solution with methanol to give

six different concentrations within the ranges:

a) 3.8-45.6 μg/ml

b) 2.1- 6.7μg/ml

c) 3.2-36.4 μg/ml

d) 4.5-51.7 μg/ml

e) 2.7-29.2 μg/ml

f) 0.58-17.3 μg/ml;

g) 2.5-18.9 μg/ml

h) 4.3-48.9 μg/ml

i) 3.3-41.8μg/ml

j) 12.7-29.2 μg/ml

k) 10.58-17.3 μg/ml

l) 12.5-18.9 μg/ml;

m) 14.3-28.9 μg/ml

n) 33.3-41.8μg/ml

o) 22.7-29.2 μg/ml

p) 12.58-17.3 μg/ml

q) 2.5-18.9 μg/ml

r) 44.3-48.9 μg/ml

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Averrhoa carambola, L. leaf extract
For calibration curves. The standard solutions were filtered through

a 0.45 μm membrane prior to injection. The standard stock and working

solutions were stored at 4°C.

Preparation of sample solutions

The extract sample of Averrhova carambola under optimized

conditions (extraction pressure: 30 Mpa; extraction temperature: 35ºC;

extraction time: one hour; 20 ml 95% ethanol modifier) was used.

After evaporating ethanol to dryness by a rotary evaporator, residue

was dissolved in methanol in a 25 ml flask, and then filtrated through a

0.45 micro-m millipore filter before HPLC injection. Three aliquots of

the solution (20 μl) were injected to RP-HPLCDAD system. The graph

obtained after the elution of compounds was compared with the standard

graph and major compounds were detected.

3.3.3. Quantitative analysis

Determination of moisture content

5 g of material was taken in a preweighed petridish. The petridish

was placed without lid into an oven at 1100C for 3 hours. The petridish

was taken out and closed immediately with alid. The dish was then

cooled in a dessicator and weighed. The amount of moisture was

calculated from the difference in weight.

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Averrhoa carambola, L. leaf extract
Estimation of Total Carbohydrate

Weighed amount of fresh tissue was homogenized with distilled

water. The homogenate was filtered using a two layered cheese cloth. The

filtrate was then centrifuged at 10,000 rpm for fifteen minutes. The

supernatant was collected and the volume was made up to 25 mL using

distilled water. An aliquot of sample was pipette out and 4 mL Anthrone

reagent was added. It was then kept in a boiling water bath for ten

minutes. The tubes were cooled and the absorbance was measured at 530

nm in a spectrophotometer. The amount of total carbohydrates was

determined using the standard graph of glucose (Hedge.J.E., et al., 1962).

Estimation of Protein

Total protein present was estimated by Lowry method. (Lowry.

O.H., et al., 1951).one gram of fresh leaf tissue was ground thoroughly in

10 mL Phosphate Buffer. The homogenate was filtered through a double

layered cheese cloth and centrifuged at 5,000 rpm for 10 minutes. The

supernatant collected was made to a known volume of 10 mL using

Phosphate buffer. An aliquot of one mL was taken and 10%

TriChloroAcetic acid (TCA) was added and shaken well. This was kept

under refrigeration for fifteen minutes and then centrifuged at 10,000 rpm

for ten minutes. The upper layer was decanted; the pellet collected and

dissolved in 10 mL 0.1 N NaOH solution. 0.1 mL aliquot was pipette out

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Averrhoa carambola, L. leaf extract
and made up to 1mL by using 0.1 N NaOH. To this 5 mL of reagent C

(mixture of 2g Na2CO3 in20 mL 0.1N NaOH and 0.25 g

KNaC4H4O6·4H2Oin 25 mL distilled water in 50:1 ratio) was added and

kept for ten minutes. Then add 0.5 mL of alkaline Folin’s reagent and

kept for thirty minutes. Absorbance was measured at 670 nm. The protein

present was calculated from the standard graph of Bovine Serum

Albumin (BSA).

Estimation of Total Phenols

Weighed amount of fresh tissue was chopped and putinboiling

methanol (80%) for ten minutes and refluxed. The refluxed matter was

homogenized. The homogenate was filtered and centrifuged at 10,000

rpm for ten minutes. The supernatant was collected and the volume was

made upto 20 mL using 80% methanol. 0.5 mL Folin-Ciocalteau reagent

followed by 2 mL 20% Sodium carbonate was added to an aliquot of

sample and mixed thoroughly. The reaction of phenol with

phosphomolybdic acid in Folin-Ciocalteau reagent in alkaline medium

produced a blue coloured complex. The tube was kept in a boiling water

bath for 1 minute, cooled, centrifuged and the supernatant was taken to

measure the absorbance at 650 nm. The amount of Total Phenols was

determined using a standard graph of catechol (Malick.C.P., et al., 1980).

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Averrhoa carambola, L. leaf extract
Estimation of Reducing sugars

1 g of fresh leaf tissue was taken and homogenized in 10 mL

distilled water. The homogenate was filtered using a cheese cloth. The

filtrate was centrifuged at 10,000 rpm for ten minutes. The supernatant

collected was made upto a known volume of 10 mL. About 0.2 mL 0f this

was taken and made upto 1 mL using distilled water. 2 mL DNS

(DiNitroSalicylic Acid) reagent was added and kept in a boiling water

bath for ten minutes. This was later cooled to get an orange red colored

solution. Absorbance was measured at 540 nm (Plummer, D.T., et al.,

1987).

Estimation of Amino acids

Fresh tissue was weighed to 1 g and refluxed in 80% methanol for

ten minutes. This was grinded thoroughly in a mortar and pestle and

filtered out. The filtrate was centrifuged at 7500 rpm for ten minutes. The

collected supernatant was made up to a known volume of 10 ml using

80% methanol. 0.1 mL of this was taken and 5 mL of Ninhydrin reagent

was added. It was shaken well and heated in a boiling water bath for ten

minutes. The tubes were cooled under running tape water and the

absorbance was measured at 570 nm. The amount of amino acids was

estimated using the standard graph of leucin (Moose. S.,et al., 1948).

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Averrhoa carambola, L. leaf extract
Estimation of Chlorophylls

Ig of fresh leaf tissue was grinded in 10 mL 80% acetone. The

filtrate obtained through cheese cloth was centrifuged at 5,000 rpm for

five minutes. The supernatant was collected and made upto a known

volume of 10 mL. From this 1 ml was taken and made upto 5 mL using

80% acetone. The absorbance was measured at 645, 663, 652 and 490 nm

for calculating the amount of Chlorophyll a, b, total Chlorophyll and

Caroteinods respectively (Arnon, D.I, 1949).

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Averrhoa carambola, L. leaf extract
3.4. ANTHELMINTIC ASSAY

The Assay was performed in Pheretima posthuma due to its

anatomical and physiological resemblance with the intestinal round worm

(Ascaris lumbricoides) parasite of human beings (Thorn, G.W., et al.,

1977).

3.4.1. Worm collection

The Indian Earthworm Pheretima posthuma (Annelida) was

collected from Botanical garden, Department of Botany, University

College, Thiruvananthapuram.

3.4.2. Anthelmintic activity

The anthelmintic assay was carried as per the method of

Anisha.N.Shah et al., 2011 with necessary modifications. The leaves of

Averrhoa carambola were dried, ground to coarse powder and boiled.

The aqueous extract obtained was filtered and concentrated by

evaporation to get extract.

3.4.3. Preparation of test sample

Samples for in vitro study were prepared by dissolving 2.5 g of

extract in 25 mL of distilled water to obtain stock solution of 100 mg/mL.

From this stock solution, different working dilutions were prepared to get

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Averrhoa carambola, L. leaf extract
a concentration range of 10, 50 and 100 mg/mL.

50 mL formulations containing three different concentrations each

of extract (10, 50 and 100 mg/mL in distilled water) were prepared and

two worms 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 of death of worms was recorded after

ascertaining that the worms neither moved when shaken vigorously or

when dipped in warm water (500C). Albendazole (10, 50 and 100 mg/mL)

was used as reference standard while distilled water as the control.

3.5. ANTIMICROBIAL STUDIES

3.5.1. Sterilization of glass wares

The petridishes, test tubes and other glasswares were washed with

a mild detergent and rinsed with sterile distilled water. Then they are

autoclaved at 1210C for ten minutes.

3.5.2. Preparation of Peptone water

The media was prepared by taking 13.6 g of Peptone powder in

1000 mL distilled water. Then it is poured in 5-6 mL in each test tube.

The test tubes were then autoclaved at 121 0C for twenty minutes and

stored.

28
Averrhoa carambola, L. leaf extract
3.5.3. Preparation of media for culturing bacteria

The media used for culturing bacteria was nutrient agar medium. It

was prepared by adding 28 g of nutrient agar in 1000 mL of distilled

water taken in a beaker. For solidification of the medium, 10 g of agar

was dissolved by placing it in a boiling water bath. Then it was

autoclaved at 1200C for fifteen minutes. Required volume of molten

medium were poured into sterile petridishes under aseptic condition and

stored. The bacterial strains used were E.coli, Enterococcus sps.,

Staphylococcus and Bacillus pyocyaneus. The pure samples were

obtained from the Department of Microbiology, Govt. Medical College,

Thiruvananthapuram.

3.5.4. Inoculation of bacteria

Inoculation was done under flame in highly aseptic condition under

laminar airflow chamber. The pure culture obtained from laboratory was

taken and from that 1-3 spores of bacteria were transferred to 5 mL of

Peptone water in the test tube. Then it was incubated for one hour at 370C

for regeneration. After one hour, this was taken out and swabbed into the

nutrient agar plate. Then it was kept aside.

3.5.5. Preparation of Disc for inhibition studies

In the disc diffusion method, discs of 4 mm diameter were cut out

from Whatman No.42 filter paper. They were sterilized by autoclaving at

1200C for fifteen minutes. Then it was stored in aseptic condition in a test

29
Averrhoa carambola, L. leaf extract
tube. During the time of treatment, the discs were taken out from the test

tube with the help of a foreceps. The crude extract of different

concentrations were pipetted out and poured into different sterile

petridish. The concentrations were 1mg/mL, 2mg/mL and 3 mg/mL of

each extract. Then 10 filter paper discs were placed into each petridish.

Cover it with a glass lid and kept for one hour so that the filter paper will

be fully saturated with crude extract.

With the help of forceps, one disc from each petridish of various

concentrations, which are saturated with crude extract, was taken. These

discs were placed on each petridish containing bacterial spores. A control

disc was treated with the solvent, methanol. The standard disc used here

is an antibacterial drug, Ampicillin. Then it was covered with petriplate

and was kept for incubation overnight at 370C. After one day, zone of

inhibition was noted only if the plant extract possess antibacterial activity

(Barry, A.L., 1980).

30
Averrhoa carambola, L. leaf extract
RESULTS AND DISCUSSION

4.1. PHYTOCHEMICAL SCREENING

4.1.1. Qualitative analysis

The qualitative analysis of leaf extract indicated the presence of

tannin, reducing sugar, Terpinoids, caumarin and saponin. But

glycosides, alkaloids, steroids and flavonoids are absent (Table 1).

4.1.2. Quantitative analysis

The qualitative analysis of fresh leaves of Averrhoa carambola, L.

showed the presence of Protein, Phenol, Amino acids, Reducing sugar,

Carbohydrate, Chlorophyll a, Chlorophyll b and caroteinods in different

amounts.

Proteins and amino acids are present in 6.92 and .078 mg/mL while

carbohydrate and reducing sugar are present in 147 and .029 mg/mL

respectively.

The pigments chlorophyll and caroteinoid are present in 4.55 and

0.015 mg/mL respectively. Among Chlorophylls, Chlorophyll a and

chlorophyll b are present in 3.64 and 3.19 mg/mL respectively.

31
Averrhoa carambola, L. leaf extract

Table.1

Name of compound Present/absent

Reducing sugar Present


Glycoside Absent
Alkaloid Absent
Terpinoids Present
Steroids Absent
Flavonoid Absent
Tannin Present
Caumarin Present
Saponin Present

32
Averrhoa carambola, L. leaf extract

Table. 2

Name of compound Amount (Mg/gL)

Protein 6.92
Phenol 0.005
Amino acids 0.078
Reducing sugar 0.029
Carbohydrate 147.0
Total chlorophyll 4.55
Chlorophyll a 3.64
Chlorophyll b 3.19
caroteinoid 0.015
moisture content 90.76%

33
Averrhoa carambola, L. leaf extract
The moisture content is about 90.76% and the amount of

Carbohydrate is the most and that of phenol is the least (Table 2).

Carbohydrates represent an important class of naturally occurring

substance. About 80% of the dry weight of green plants is made of

carbohydrates. Catabolism of carbohydrates provides the major share of

the energy requirement for life and performance of work, glucose is an

immediate source of energy in organism (Lehninger, 1995)

Proteins are the essential constituents of life which make up about

12% of protoplasm. They form enzymes, transport materials, regulation

of metabolism, movement and defense reactions (Lehninger, 1995)

Phenols are biologically active compounds. They are aromatic

compounds possessing one or more phenolic carboxyl group. Plants

respond to infection or injury by the accumulation of phenols. Phenols are

active against a wide range of microorganisms. It form the basic frame

work of lignin and also helps in photosynthesis by absorbing U.V. passed

on to the chlorophyll (Takayama, et al., 2001)

Chlorophyll and Caroteinoid are the major photosynthetic pigments

found in plants. Chlorophyll plays crucial role in the phenomenon of

photosynthesis. Chlorophyll a and Chlorophyll b are the common

chlorophyll pigments in green plants. Caroteinoids include Carotenes and

xanthophylls (Arnon, 1949).

34
Averrhoa carambola, L. leaf extract
4.1.3. Identification of compounds by HPLC

The HPLC method is adopted for the identification of compounds

present in the Methanolic extract of leaves. The compounds identified

were listed below (List.1). About 13 compounds were identified.

35
Averrhoa carambola, L. leaf extract
LIST-1

HPLC Compounds Identified – Averrhova

1. Gallic acid

2. Citric Acid

3. Epigallocatechin

4. Epigallo Catechin Gallate

5. Catechin

6. Epicatechin

7. L-Ascorbic Acid

8. Proanthocyanidin

9. Abcisic Acid

10. Vomifoliol

11. Roseoside

12. Methyl benzoate

36
Averrhoa carambola, L. leaf extract
4.2. Anthelmintic assay

The leaves of Averrhoa carambola displayed a significant

anthelmintic activity in dose depended manner giving shortest time of

paralysis (P) and death (D) with100mg/ml concentration as shown in

Table 3 (Fig 5, 6 and 7). The anthelmintic activity of aqueous extract was

comparable with that of standard drug at 100mg/ml concentration, plant

extract showed paralysis in 9.44 minutes and death in 16 minutes while

Albendazole exhibited similar effects at 8 and 18 minutes respectively

(Fig.7). Control worms were live up to 24 hours (Fig.8).

As the plant extracts are found to contain tannins, which are

Polyphenolic compounds, there is a possibility that their effects are like

synthetic phenolic anthelmintics e.g. - niclosamide, oxyclozanide,

bithionol etc. which are reported to interfere with energy generation in

helminth parasites by uncoupling oxidative phosphorylation(Martin, R.J,

1997). Another possible anthelmintic activity of tannins is that they can

bind to free proteins in gastrointestinal tract of host animal or

glycoprotein on the cuticle of parasite and may cause death(Thompson,

D.P. et al., 1995).

37
Averrhoa carambola, L. leaf extract
Table 3

Anthelmintic activity of Averrhoa carambola,L.

Time taken for Paralysis (P) and


Concentration Death (D) of Pheretima posthuma
Sample
(mg/ml) in minutes

P D
Albendazole 10 28.23±1.34 67.36±5.65
Albendazole 50 13.36±1.46 38.5±1.32
Albendazole 100 8.4±1.14 17.48±2.42
Plant extract 10 83.05±8.43 187.5±30.01

Plant extract 50 25.55±2.54 33.12±3.15

Plant extract 100 9.44±2.01 17.24±1.47

All values represent Mean ± SEM; n=2 in each group.

38
Averrhoa carambola, L. leaf extract
4.3. Antibacterial assay
The present study indicates that the extract from leaves showed

positive reaction against pathogens. E.coli, Enterococcus sps.,

Staphylococcus and Bacillus pyocyaneus.

In the present investigation, the soaked filter paper discs carrying

plant extract in methanol bring about an zone of inhibition of 1.9 mm,

2.1mm and 1.0 mm for 100mg/mL for E.coli , Enterococci sp. and

Bacillus pyocyaneus respectively. The zone of inhibition for 50 mg/mL

were 1.7 mm, 1.6 mm, 1.5mm, 1.9 mm for E.coli , Staphylococcus sp..,

Enterococci sp. and Bacillus pyocyaneus respectively. Similarly 1.5mm

and 1.6 mm were the zone of inhibition for E.coli , and Enterococci sp.

(Fig.9, 10,11 and 12), (Table.4).

The cell walls of gram negative bacteria were thinner compared to

that of gram positive bacteria. So they undergo lysis more easily than the

others. This makes them more sensitive to drugs than gram positive

bacteria.

The immediate reason for positive results may be due to the

presence of chelating agents and also due to the favorable change in the

permeability of cell membranes.

39
Averrhoa carambola, L. leaf extract

Table.4

Zone of inhibition of bacteria

Test organism Disc Conc.of Zone of Control Standard


no. extract inhibition (Methanol) (mm)
(mg/mL) (mm)
E.coli 1 100 1.9
2 50 1.7 - 6
3 10 1.5
Staphylococcus sp. 1 100 -
2 50 1.6 - 4.7
3 10 -
Enterococci sp. 1 100 2.1
2 50 1.9 - 3.6
3 10 1.6
Bacillus pyocyaneus 1 100 1.0
2 50 1.5 - 3.9
3 10 -

40
Averrhoa carambola, L. leaf extract
Thus from the present study, it was proved that Averrhoa

carambola has antibacterial property as found in previous works

(Md.Masum Mia, et al., 2007; Burgorn Sripanidkulchai. et al., 2002)

41
Averrhoa carambola, L. leaf extract
SUMMARY

Averrhoa carambola, L.of Family Geraniaceae is a tree widely

cultivated in Asian and African countries. The leaves were used for the

treatment of biliousness and diarrhea.

Leaves collected from Thiruvananthapuram were used to make

extract by using distillation method using soxhelet apparatus with

methanol as solvent. The weighed shade dried and powdered material

was continuously heated at the boiling point of solvent. Later it was dried

and transferred to a pre-weighed sample bottle.

Different biochemical parameters like reducing sugar, flavonoids,

steroids and terpenoids, tannins, caumarins, saponins and glycosides were

analysed. Quantitative test for plant like determination of moisture, total

carbohydrate, aminoacid, total protein, total phenol and chlorophyll were

done using standard methods.

The anthelmintic assay was done in Pheretima posthuma. Averrhoa

carambola showed anthelmintic activity. However dose and form in

which they can be used requires standardization. . Future scope involves

isolation of phytoconstituents responsible for activity. Pure cultures of

Gram positive - Enterococci sps. and Staphylococcus sps. And gram

negative- E.coli and Bacillus pyocyaneus were used for antibacterial

42
Averrhoa carambola, L. leaf extract
studies and zone of inhibition were observed high in crude extract

(100mg/mL).

Thus from the present study, it was made clear that Averrhoa

carambola, L. is not only a food source, but also possess very effective

anthelmintic and antibacterial properties. Thus the economic potentialities

of this plant may be exploited in future, after more extensive studies.

43
Averrhoa carambola, L. leaf extract
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