Identification of Soursop Seeds (Annona Muricata L.)Extract as A ...

ISBN : 978-602-72418-2-4

International Seminar Pokjanas TOI

Faculty of Pharmacy Pancasila University 74

Identification of Soursop Seeds (Annona Muricata L.)Extract as A Candidate

Against The Aedes Aegypti L. Musquito

Vector Control DBD

SARAH ZAIDAN, RATNA DJAMIL, SITI NURAINI

Faculty of Pharmacy, Pancasila University

Jln. Srengseng Sawah Jagakarsa, Pasar Minggu, Jakarta Selatan 12640.

Abstract: Aedes aegypti L. mosquitos are the disease vectors of dangue hemorrhagie fever (DHF),

caused by dengue virus which is transmitted by Ae. Aegypti mosquito. The effort to control Ae. aegypti

vector have been done in so many times, including chemical, physical, and biological control method.

Multilevel extraction by kinetic maceration have been done with soursop seeds (Annona muricata L.)

with the solvent of n-hexane, ethyl acetate and 70% of ethanol. Subsequently, the obtained extract is

tested phytochemical screening along with the powder and larvicidal activity against Ae. aegypti. The

results of phytochemical screening of the powder and 70% ethanol extract of soursop seeds have

obtained the compound of saponin, triterpenoid and coumarin. In the n-hexane extract have obtained

triterpenoids and in ethyl acetate extract which is found triterpenoids and coumarin. Based on the activity

test against the larva of Ae. aegypti from n-hexane extract, ethyl acetate and ethanol 70% of soursop seeds

sequentially, show LC50 values were about 198,610 ppm, 74,798 ppm and 67,042 ppm. Soursop seeds

extract that has the highest activity is 70% ethanol extract. These are indicate that the chemical

compounds which is found in soursop seeds have a potential as a larvicides.

Keywords:Aedes aegyti L., Annona muricata L., soursop seeds, larvasida.

INTRODUCTION

Mosquitoes are insects that are often found in tropical countries such as Indonesia. Besides

disturbing human life, the presence of mosquitoes act as vectors of some diseases. In Indonesia, a disease

transmitted by mosquitoes is still a health problem because of the high mortality rate caused. Some

diseases transmitted by mosquito vectors such as filariasis, malaria and dengue fever (DBD)(1,2)

. Aedes

aegypti L. mosquitoes are diurnal, or active during the morning and afternoon. Ae. aegypti mosquitoes

carrying dengue virus causes dengue which is obtained from infected individuals and multiply in the body

and the salivary glands of mosquitoes (2)

.

Dengue disease not only in children but in all ages. DBD becoming known in Indonesia in 1968 in

Surabaya and Jakarta, and then continue to expand as the spread of dengue endemic area. The number of

cases of dengue and widely spread is increasing along with the increasing mobility and population

density. There are 150,000 cases of dengue in 2007 and continued to increase until 2010. In addition,

WHO reported more than 35% of the population living in urban areas affected by the disease. Until now

there is no specific vaccine to treat dengue fever, and the only control vector for controlling the spread of

the disease(2, 3, 4, 5)

.

Vector control mosquitoes until today, still put emphasis on the use of insecticides, for example, is a

synthetic larviciding. Larvicides in general have a higher efficacy and the results can be seen quickly. The

use of continuous and repetitive can cause environmental pollution and resistance against the target

organism. This encourages biological larviciding as controlling mosquito vectors (Biolarvasida). These

biological larvicides are safer for humans, readily available and environmentally friendly(2, 6)

.

Biological larvicides useful for the improvement of local natural resources. Local plant as a potential

biological larvicides generally from families Annonaceae, including soursop (Annona muricata L.).

Empirically, it has been much research done on the soursop as larvicides. The plant parts are potentially

as larvicides are seed (semen)(7)

. Soursop seed (with shell beans) have larvicidal activity against Ae.

aegypti with LC50 value of 244.27 ppm for the ethanol extract of the seeds of soursop6, 8

. In addition,

Ward et.all, reported that seeds of soursop and sugar apple seed (without seed coat) effect on mortality

Chrysomya bezziana fly larvae(9, 10)

.

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The main active compounds from the seeds of the soursop is annonacin and squamocin belonging

asetogenin compound. Squamocin annonacin compound of the family Annonaceae and is reported to

have toxicity properties are quite effective against insects of the order Diptera (Ae.aegypti L.) which are

cytotoxic, and neurotoxic. Asetogenin compounds can inhibit the action of the enzyme NADH in the

mitochondria, causing the death of larvae, as well as toxic contact and stomach poison to insects(6, 9, 10)

.

This research is continuing efforts to obtain biological larvicides. In this study, conducted Qualitative

identification through screening phytochemicals and seen the activity of seed extracts of soursop (Annona

muricata L.) (without seed coat) against Ae. aegypti. Extract obtained from extraction method using a

multi-storey penyari with different polarities ie n-hexane, ethyl acetate and ethanol 70% and seen the

highest larvicidal activity of the three fractions of the extract. Extract larvicidal activity test using

Standard Methods of Pesticide Efficacy Testing Households and Vector Control11

. The data seen LC50

values were obtained by probit analysis using Probit Analysis Program Epa Used For Calculating LC /

EC Values Version 1.5.

MATERIAL AND METHODS

MATERIAL. Soursop seeds (Annona muricata L.) were obtained from Balitro (Research Institute for

Spices and Medicinal Plants) Cimanggis, Bogor. Determination at Bogoriense Herbarium, Research

Center, LIPI Cibinong , Bogor.

Extraction. Seed Soursop (Annona muricata L.) which have been dried in the sun was directly

crushed and blended into a fine powder. Powdered crude drug was extracted by maceration kinetic in

stages using different solvent polarity is n-hexane, ethyl acetate, and ethanol 70% at room temperature

until the extracted perfectly, then filtered with cotton and proceed with filter paper, pulp, and each extract

n -heksan, ethyl acetate, and ethanol is 70% separated. Each extract was concentrated by vacuum rotary

evaporator at a temperature of 450 C to obtain a viscous extract n-hexane, ethyl acetate and ethanol 70%.

Identification with phytochemical screening. Phytochemical screening performed on pollen and

seed extract of soursop with Farnsworth method in Biological and phytochemical screening of Plant seed

sirsakdilakukan to identify the qualitative content of secondary metabolites in seed soursop.

Flavonoids. 2 grams of powder simplisia or 0,67 g of n-hexane extract and ethyl acetate extract;

0.15g of extract ethanol 70% boil with 100 ml of hot water for 5 minutes, then filtered with filter paper, 5

mL filtrate of extract solution coupled with a bit of powdered zinc or magnesium and 1 mL of 2 N HCl

and 5 mL amyl alcohol . Flavonoids compounds would pose orange to red(12)

.

Saponins. Entering 10 ml sample into a test tube and shake for 30 seconds and observe what

happens. If the foam is formed solid (not lost for 30 seconds) the identification showed the presence of

saponins(12)

.

Coumarin. 2.12 grams of powder simplisia or 0.67 g of n-hexane extract and ethyl acetate extract;

0.15g of extract ethanol 70% included in the test tube and add 10 ml of chloroform, heated 20 minutes on

waterbath is then cooled. After it is filtered with filter paper, the filtrate waterbath until dry. The residue

was added 10 mL of hot water, then cooled and put into a test tube, add 0.5 mL of 10 % ammonia

solution and then observed under UV light at a wavelength of 365 nm (blue or green fluorescence showed

the presence of cumarin(12)

.

Volatile oil. 2 of powder simplicia and 0.67 g extract put into a test tube, then added 10 mL of

petroleum ether, at the mouth of the tube fitted with a mouthpiece that has given cotton that has been

moistened with water, then heated above waterbath10 minutes after the cold water and filtered with a

filter paper. The Obtained filtrate is evaporated in the vaporizer cup, the residue is dissolved in 5 mL

ethanol and then filtered with filter paper. If residues smelling aromatic indicate a of compounds volatile

oils(12)

.

Kuinon. 5 ml of solution experiments inserted into a test tube, add a few drops of 1 N sodium

hydroxide solution, Occurs in red indicate a compounds of quinine(12)

.

Steroids/Triterpenes. 1.10 grams of powder or soursop seed extract: 0.33 g extract of n-hexane;

0.34 g of ethyl acetate extract; 0.67 g of ethanol extract 70% extract, macerated with 20 mL eter for 2

hours, then filtrated the solution, and A total of 5 mL of the extract solution evaporated to dryness, then

added with a reagent Lieberman- Burchard. green - red color arising indicates compounds terpenoids or

steroids(12)

.

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Tannin. 2 grams of powder simplisia or 0.67 g of n-hexane extract and ethyl acetate extract; 0.15g

of extract ethanol 70% added 100 mL of water, boil for 15 Minutes, cooled and filtered. divided to each 5

mL filtrate (reaction tubes) : Added a few drops of solution of iron (III) chloride 1 %, Changes blue or

blackish green and Added a few drops of 1 % solution of gelatin to form white precipitate indicates the

compounds of tannins . To 5 mL Second filtrate was added 15 mL reagent Stiasny (formaldehyde 30% -

hydrochloric acid = 2:1), the precipitate formed pink color indicates the presence of tannins katekuat.

Subsequently the precipitate is filtered, the filtrate who saturated with sodium acetate powder, add a few

drops of solution of iron (III) chloride 1 %, occurred in blue ink Showed the presence of tannins galat(12)

.

Alkaloids. 2.12 grams of powder simplisia or 0.67 g of n-hexane extract and ethyl acetate extract;

0.15g of extract ethanol 70% is inserted in a porcelain bowl and then add 5 mL of ammonia 30% crushed

and then added 20 mL chloroform and crushed again, then filtered. The filtrate obtained was added HCl 1

N as much as 5 ml and then separated into 2 sections namely A and B. The filtrate A coupled with Mayer

reagent, filtrate B coupled with Dragrendroff reagent. With reagent Meyer gives a white precipitate, and

Dragendorff reagent give an red brick precipitate(12)

.

Larvasidal activity test. Larvae Maintenance. Mosquito eggs incubated in a plastic container

(tray) measuring 20 x 15 x 10 cm3yang containing distilled water. The eggs will hatch within 24 hours of

becoming the first instar larvae, then the 2nd day will have become instar II stage of development, at this

stage larvae fed chicken liver, then after 1-2 days will be changed again to the third instar.

Implementation of Experimental Test larvicidal activity. Larvicidal activity test was conducted

using ”Pesticide Efficacy Testing Standards Household and Vector Control”. Carefully weigh

approximately 100 mg extract and then dissolved in 100 mL of of solvent. This solution is a mother liquor

(1000 ppm). The mother liquor 18.750 ml pipette; 12.500 mL; 6.250 mL; 3.125 mL; 1.250 mL

respectively inserted into plastic cups that have ditara 25 mL to obtain a concentration of 750 ppm, 500

ppm, 250 ppm, 125 ppm, 50 ppm, then evaporated completely. Each concentration was made in 3 plastic

cups (triplo), then into individual plastic cups partially added to 25 mL of distilled water homogenkan,

and included 20 third instar larvae of Ae. aegypti. Observations were made after 24 hours of exposure to

the test solution and counted the number of larvae were dead and stated in the presentation of death.

Negative controls only solvent without the extract, in the same way. Positive controls carried out on

Temephos 1 ppm.

Data Processing Methods. Test data processing is done systematically using probit analysis

method. Probit analysis is used to determine the percentage of larval mortality LC50 of Ae. aegypti L. uses

Epa Probit Analysis Program Used For Calculating LC/EC Values Version 1.5. In Epa Probit

Analysis Program Used For Calculating LC/EC Values Version 1.5. the data entered is the relationship

with the concentration of the value of the average percentage mortality of larvae of Ae. aegypti.

RESULT AND DISCUSSION

Phytochemical Screening. The phytochemical screening via Farnsworth method was conducted

using powder of simplicia and Soursop seeds and sugar-apple seeds extract. In powder and extract

having metabolite compound such as saponin, triterpenoid, and cumarin. The result of phytochemical

test is shown in Table 1. Table 1. Result of phytochemical screening of Soursop seeds (Annona muricata L.) and sugar-

apple seeds (Annona squamosa L.) powder and extract.

Notes : + = giving positive reaction − = giving negative reaction

No Secondary

Metabolites

Simplicia

powder

Ethanol 70%

Extract

Ethyl acetate

Extract

N-heksan

Extract

1. Alkaloids - - - -

2. Flavonoids - - - -

3. Saponins + + - -

4. Kuinon - - - -

5. Tannin - - - -

6. Steroids /

triterpenoids

- /+ - / + - / + - / +

7. Volatile oil - - - -

8. Coumarin + + + -

ISBN : 978-602-72418-2-4



In Table 1 it can be seen that the results of the qualitative identification of secondary metabolite content

of the seed powder soursop (Annona muricata L.) by means of screening phytochemical compounds derived

class of saponins, triterpenoids, and coumarin. In the n-hexane extract obtained compound class of

triterpenoids. At the ethyl acetate extract obtained triterpenoids groups and coumarin compounds. While the

70% ethanol extract derived class compound saponin, triterpenoids, and coumarin.

Larvicidal Activity Test. Larvicidal activity test extract n-hexane, ethyl acetate, and ethanol 70% seed

soursop done by the method of “Pesticide Efficacy Testing Standards Household and Vector Control” for

mosquito larvae Ae.aegypti L. larvae used test is the third instar larvae of mosquitoes Ae.aegypti because has

a fairly good resistance against external environment and durability stronger mechanically when the transfer

of the larvae, and have a long time to turn into adult mosquitoes. Test solution at a concentration of 50, 125,

250, 500 and 750 ppm generated triplo, then put 20 third instar larvae of Ae. aegypti L. and counted the

number of larvae mortality after 24 hours of observation. Negative controls only the solvent used and

Temephos (larvicidal commonly used) as a positive control.

Table 2. The average percentage mortality of larvae of Ae. aegyptiL. extract after exposure to n-hexane, ethyl

acetate and ethanol 70% soursop seeds on a 24-hour observation.

The results showed that the larvae of Ae. aegypti exposed seed extract of soursop (without skin) for 24

hours to come LC50 ie, n-hexane extract of soursop seeds of 198.610 ppm, ethyl acetate extract had LC50

values of 74.798 ppm and 70% ethanol extract of the seeds of the soursop has a value of 67.042 ppm LC50.

This shows that the 70% ethanol extract of soursop seed having the highest activity as larvicides. This study

can be interpreted that the seed extract of soursop (without skin) also has larvicidal activity. This can be

caused by secondary metabolites contained in the soursop seed saponin, coumarin suspected triterepenoid and

potentially as larvasida(13)

.

Saponins allegedly able to diffuse into the cuticle layer of larvae that can damage cell membranes and

toxic compounds can be entered and off the larvae. Saponins have a bitter taste and sharp and can cause

irritation of the stomach. Larvae digestive tract, particularly the midgut (midgut) is the major site of

absorption of nutrients and digestive enzymes seksresi. Saponin absorption into the intestine larvae can

inhibit the action of digestive enzymes and cause damage to the cells in the channel pencernaan(14)

.

Triterpenoids also thought to be as antifeedant on the larvae so that the larvae loss of appetite, this led to

the loss of energy and development of larvae will be hampered even can cause mortality15

. In addition,

coumarin is also reported as larvicides because potentially able to change the detoxification ability to

reversibly or irreversibly inhibit the enzyme cytochrome P45016

.Dari third ability of secondary metabolites in

seed soursop concluded that sugar apple seeds potentially sebagaii larvicides against mosquito larvae Ae.

aegypti L.

Mortality of larvae on seed extract of soursop seeds (Annona muricata L.) allegedly also because of the

effects of the component compounds acetogenin toxic squamosin contact. Where after the larvae exposed to

Concentration

(ppm)

% Mortality

Type Solvent Control

n-hexane Ethyl acetate Ethanol 70% Negative

(Solvent)

Positive

(Temephos 1 ppm)

750 93.35 95 100 0 100

500 78.35 88.35 100 0 100

250 50 73.35 98,35 0 100

125 20 65 58,35 0 100

50 15 40 45 0 100

LC50 (ppm) 198.610 74.798 67,042 - -

Linear

regression

a = -115.7371

b = 70.9888

r = 0.9654

a = -34.9054

b = 45.5671

r = 0.9926

a = -43.2775

b = 52.5234

r = 0.9302

-

-

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the extract, the compound into the body of Ae. aegypti through physical contact and the case of death of the

larva. Prijono (1994) in Ward et.al (2005) states that the absorption of toxic insecticides contact occurs

largely in the cuticle. Active compounds will penetrate into the insect's body through the part that is covered

by a thin cuticle, such as membrane between segments. The ability of the compound asetogenin stomach

poison works by absorption seyawa on soursop seed extract into the wall fosfolirasi larvae and able to inhibit

oxidative chain so that the cell respiration is inhibited activity of Ae. aegypti because of breathing stopped.

Squamocin compounds in the seeds of soursop allegedly able to diffuse from the thin cuticle layer to spread

throughout the body Ae. Aegypti through hemolimfa flow(17)

.

Mortaitas larvae of Ae. aegypti showed signs as follows: larvae do not move when touched, bodies pale

white larvae, elongated body shape or kaku1. The color can be seen more clearly with the aid of a stereo

microscope and optilab. Differences larvae of Ae. aegypti normal and who have died can be seen in Figure 1.

.

Figure 1. The third instar larvae of Ae. aegypti normal (A); and third instar

larvae of Ae. aegypti die (B)

Figure 2. Graph average percentage mortality soursop seed extract on a 24 hour observation (x-axis:%

average mortality of larvae and the y-axis: concentration soursop seeds extract (ppm)).

A B

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From Figure 2 shows that the higher the concentration of soursop seed extract, the higher the death rate

of Ae. aegypti L. The solvent n-hexane, ethyl acetate and 70% ethanol and distilled water as a negative

control test the same activity against larvae of Ae. aegypti, and the results obtained all the larvae do not occur

death. This indicates that the solvent does not affect the mortality of larvae. Temephos as a positive control, in

which the larvicidal activity at a concentration of 1 ppm trials have demonstrated 100% mortality against

larvae of Ae. aegypti L.

CONCLUSION

Based on the results of phytochemical screening of the seeds of soursop (Annona muricata L.) obtained by

the content of secondary metabolites. The test results with the larvicidal activity and data analysis has been

done, it can be concluded that the 70% ethanol extract of the seeds of the soursop has the highest activity as

larvicides against Ae. aegypti L. with LC50 values of 97, 462 ppm.Ethanol 70% extract of the seeds of

soursop (Annona muricata L.) has a good chance to be used as biological insecticides to control mosquito

larvae that are environmentally friendly.

REFERENCES

1. Kaihena M, Vika L, Maria N. Efektivitas ekstrak etanol daun sirih (Piper betle L) terhadap mortalitas

larva nyamuk Anopheles sp dan Culex . Molluca medica. ISSN: 1979-6358

2. Susanti PD, Danang B, Dini S, Susilawati. Penggunaan ekstrak kulit kayu gemor (Nothaphoebe

coriacea K.) sebagai larvasida hayati terhadap tingkat mortalitas jentik nyamuk Aedes aegypti serta

dampaknya pada kualitas air hujan. ISSN 1978-8096. 2013;9:117–22.

3. Hadi, Upik Kesumawati. Penyakit tular vektor: demam berdarah dengue. Bogor: Fakultas

Kedokteran Hewan IPB. 2005

4. World Health Organization. Dengue: guidelines for diagnosis, trearment, prevention and control, new

edition. Swiss. 2010. h.5.

5. Palgunadi BU, Asih Rahayu. Aedes aegypti sebagai vektor penyakit demam berdarah dengue. Surabaya:

Universitas Wijaya Kusuma. 2011.

6. Rosmayanti, Kiki. Uji efektivitas ekstrak biji sirsak (Annona muricata L ) sebagai larvasida pada larva

Aedes segypti instar III/IV (skripsi). Jakarta: Fakultas Kedokteran Dan Ilmu Kesehatan Universitas Islam

Negeri Syarif Hidayatullah. 2014. 1-3, 16-17, 37-40.

7. Mulyawati AP, Hayati EK, Nashihuddin A, Tukimin. uji efektivitas dan identifikassi senyawa

ekstrak biji sirsak (Annona muricata Linn.) yang besifat bioaktif insektisida nabati terhadap hama

thrips. Alchemy 2010;2(1): 104-1575.

8. Taslimah. Uji efikasi biji srikaya (Annona squamosa L.) sebagai bioinsektisida dalam upaya integrated

vector management terhadap Aedes aegypti (skripsi). Jakarta: Fakultas Kedokteran Dan Ilmu Kesehatan

Universitas Islam Negeri Syarif Hidayatullah. 2014. h.1-6, 23-28,76-81.

9. Wardhana A.H, Amir H, Muharsini S, dan Yuningsih, Veteriner BP. Uji Keefektifan Biji Sirsak (Annona

muricata) dan Akar tuba (Derris edliptica) terhadap Larva Chrysomya bezziana secara In Vitro. 2006;

1013-1017.

10. Wardhana AH, Amir H, dan J Manurung, Veteriner BP. Uji efikasi ekstrak heksan daging biji

srikaya ( Annona squamosa L ) terhadap pertumbuhan larva lalat Chrysomya bezziana secara in

vitro. 2004. 134-142. 11. Departemen Pertanian Indonesia. Metode standar pengujian efikasi pestisida rumah tangga dan

pengendalian vektor. Direktorat Pupuk dan Pestisida dan Direktorat Jenderal Prasarana dan Sarana

Pertanian Kementrian Pertanian. 2012.h.20-23

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12. Farnsworth NR. Biological and phytochemical screening of plants. Journal of pharmaceutical, Sci.

1966. (55). No 3.

13. Riswanto S. Uji Efektivitas Pestisida Nabati terhadap Hama Spodoptera litura (Lepidoptera: Noctuidae)

pada Tanaman Tembakau (Nicotiaana tabaccum L.). Medan: Universitas Sumatera Utara. 2009.20-23.

14. Susilowati D, Rahayu MP, Prastiwi R. Efek Penolak Serangga (Insecct Repellent) dan Larvasida Ekstrak

Daun Jeruk Purut (Citrus hystrix D. C.) terhadap Aedes aegypti. Surakarta:Fakultas Farmasi, Universitas

Setia Budi.

15. Nopitasari. Uji Aktivitas Ekstrak Biji Langsat (Lansium domesticum Cor.) sebagai Larvasida Aedes

aegypti. Universitas Tanjungpura. 2013. 12-14.

16. Venugopala KN, Raquel MG, Kabange K, Bandar EA, Mahesh VA, Bharti O. Evaluation of

halogenated coumarins for antimosquito properties. Hindawi Publishing Corporation The Scientific

World Journal, Vol. 2014, 4.

17. Wardhana A.H, Amir H, dan J.Manurung, Veteriner BP. Efektifitas ekstrak biji srikaya

(Annona squamosa L ) dengan pelarut air , metanol dan heksan terhadap mortalitas larva caplak

boophilus microplus secara In Vitro. 2005.134–42.

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Faculty of Pharmacy Pancasila University

81

Antimicrobial and Biology Activity from

Extract Herbs Parasite Soursop (Dendropthoe pentandra L.)

ERLINDHA GANGGA*, LIA KARTIKA SARI

Faculty of Pharmacy Pancasila University, Jl Srengseng Sawah, Jakarta 12640.

e-mail: [email protected]

Abstract: Parasite of Soursop (Dendropthoe pentandra L.) is one of many plants that grow in

Indonesia.Indonesia is a country who has a many plants of drugs, where the plant has been used since

the time of our ancestors to cure various diseases. The parts of the plants used leaves, rods, roots or

whole plant. Besides that parts, the people also used Loranthus for medicine. Loranthus is a plant

parasite and a half parasite or substances that still have green leaves (chlorophyll) used for the

assimilation process. In addition parasite also absorb the food from its host plant, so the loranthus

has the same activity with host plants. The species of loranthus usually used is the parasite of the

species Dendropthoe pentandra which grows on a soursop. hosts. Parasite of soursop plants has

been cleaned from dust and then dried in powder. The powder and the extract herbal parasite

soursop conducted phytochemical screening.After that, did the antimicrobial activity by diffusion

methode and the biological activity test (LC50) used the BSLT method. In testing the

antimicrobial activity of Escherichia coli and Staphylococcus aureus from ethyl acetate and

methanol extracts retrived power resistor from concentration 2,43.10-3

%. The activity results of

Candida albicans from n-hexane, ethyl acetate and methanol extract had retrived inhibitory

from concentration 28.14%. The biological result used the BSLT method in soursop loranthus

showed that methanol extract has a high (LC50) activity is 21.22 ppm; 510.07 ppm from n-hexane

extract and ethyl acetate extract of 87.78 ppm.

Keyword : Herbs parasite of soursop (Dendropthie pentandra L.), biology activity methode, diffusion

methode Escherichia coli, Staphylococcus aureus ,Candida albicans

INTRODUCTION

Indonesia is a rich country of biological resources and known as one of the larger mega biodiversity in the

world. Indonesia has about 17% species from all around the world. Large tropical forest with biological

diversity is natural resources which is priceless. And known as a warehouse of herb, Indonesia has a

nickname as live laboratory. Parasite of Soursop ( Dendropthoe pentandra L.) is one of many plants

that grow in Indonesia has been used empirically as medicinal plant which is a herbaceous that can be

grown easilyon tropical land. According to literature this plant containing highly Flavonoid, saponine,

alcaloid, tanine, amino acid, lorantilalkohol, kholin, and fat.

To get the biological activity test (LC50) and antimicrobe Parasite of Soursop ( Dendropthoe

pentandra L.) using extraction was done by maceration with ethanol 70 % , and partitioned gradient

started from n – hexan, ethyl acetat, and n- buthanol . after that the extracts were done phytochemical

screening method biological activity (cytotoxic ) with BSLT method and antioxidant activity with DPPH

free radical scavenging.

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82

MATERIAL AND METHODS

MATERIAL. Parasite of Soursop ( Dendropthoe pentandra L.), n–hexan, ethyl acetat, Methanol,

shrimp larvae (Artemia salina Leach), NaCl. Escherichia coli, Staphylococcus aureus, dan Candida.

METHODS. Extraction was done by maceration with started from n–hexan, ethyl acetat, and n-

methanol . after that the extracts were done phytochemical screening method , biological activity with

BSLT method and antimicrobial activity with.

Research Stages. 1.Sample preparation 2. Extract forming and fractionation 3. Phytochemical

screening 4. Toxicity Test 5. Antimicrobial Activity Test

Extract Forming and Fractionation. As much as 300.1 g kelor leaf ( Moringa oleifera Lamk L.)

leaf powder was placed in a jar, then maceration with ethanol 70 % and partitioned gradient started from

n-hexane, saturated ethyl acetate, and n- butanol. Rotavapor was used to saturate the filtrate until thick

extract formed. The waste was then thrown.

Phytochemical Screening. Content identification of kelor leaf’s secondary metabolite was done using fresh leaf and thick extract of n-hexane, ethyl acetate, and methanol, which include: 1. Alkaloids

identification, 2. Flavonoid identification, 3. Saponin identification, 4. Tannin identification 5. Kuinon

identification 6. Steroid / triterpenoid identification 7. Aetheric oil identification 8. Coumarin

identification.

Cytotoxic Test. Writer chose BSLT (Brine Shrimp Lethality Test) method for cytotoxic test using

natural ingredients of shrimp larvae (Artemia salina Leach) and sea water as the media. All substances

were tested three times in a vial of 5mL sea water and 10 larvae. Observation was done after 24 hours, the

data was analyzed to get LC50 using probit analysis. The next step was placing ± 20mg of Artemia salina

Leach.’s egg inside the hatching container filled with synthetic sea water. The sea water was prepared by weighing 38 g salt without iodine and dissolve it with 1L of water, then filter it with Whatman paper and

radiate it with 18 watt lamp. After 24 hours, hatched egg became nauplii and relocated to other place.

After the next 24 hours, nauplii is ready to be used as a test animal.

Antimicrobial Test. Anti-microbial assay was done by diffusion using paper disc with 6 mm

diameter. The paper disc contains anti-bacterial substance and then placed in the jelly’s surface which has been inoculated and incubated at 37°C for 18-24 hours. Activities decided by the blocked zone which

formed by the clear zone around the anti-bacterial substance. Other than diffusion, this research also used

positive control chloramphenicol and amphotericin B. Chloramphenicol has anti-microbes spectrum

activities for both Gram Positive Bacteria and Gram Negative Bacteria, and has bacteriostatic

characteristic where it blocked microbes’ protein synthesis. Amphotericin B also has similar characteristics where it served as wide spectrum anti-fungus.

RESULT AND DISCUSSION

Phytochemical Screening Result. Results from the phytochemical screening powder of Parasite of

Soursop (Dendropthoe pentandra L.) and the extract n- hexan,aetyl acetat, methanol showed that the

herbs powder contained, flavonoid, saponine, tannin, quinon and steroid, where as n-hexane extract

contained steroide, and ethyl acetate extract contained, flavonoide, tannin, steroide,. Furthermore,

methanol extract contained flavonoide, saponine, tannin and quinon

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83

Table 1. Phytochemical screening result.

Table 2. Cytotoxic test by BSLT method.

Ekstrak Nilai IC50 ( bpj )

Table 3. Antimicrobial activity.

CONCLUSION

1. Flavonoid, saponin, tannine, steroid and coumarin compound were found from phytochemical

screening of herbs powder and Parasite of Soursop (Dendropthoe pentandra L.).

2. Cytotoxic test by BSLT method found that thick methanol extract has the highest toxicity level of

LC50 21.22 ppm, ethyl acetate extract of 87.78 ppm and n-hexane extract of 501, 07 ppm,

3. Antimicrobe activity test found that methanol extract has, ethyl acetate extract has activities to

Escherichia coli, Staphylococcus aureus, and Candida albicans and has anti-microbes spectrum

activities for both Gram Positive Bacteria and Gram Negative Bacteria, and anti fungus (broad

spectrum ), n-hexane extract has activities to Candida albicans and has anti microbes/ anti-fungus

only.

No Compound Herbs Powder n- Hexane Ethyl acetate Methanol

1. Alkaloid - - = -

2 Flavanoid + - + +

3 Saponine + - - +

4 Tannine +/- - +/- +/-

5 Quinon + - - +

6 Steroide/

triterpenoide +/- +/- +/- +/-

7 Volatile oil - - - -

8 Coumarine - - - -

E

Extract

LC 50

Value( ppm)

n- Heksan

510.07

Ethyl Acetat 87.78

Methanol 21.22

NO

Extract

Microbe

Escherichia colli Staphylococcus aureus Candida albicans

1 n- Hexane Negative Negative Positive

2 Ethyl acetate Positive Positive Positive

3 Methanol Positive Positive Positive

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84

REFERENCES

1. Meyer BN, et. al. Brine shrimp: a convenient general bioassay for active plant constituens. Planta

medica. Volume 45. 1982. h. 31-4.

2. Famsworth, NR. Biological and phytochemical screening of plant 55(3). J Pharm. Sci;

Chicago. 1985. h. 28-58.

3. Jawetz E, Melnick JL, Adelberg EA. Mikrobiolog kedokteran. Edisi 20. Alih bahasa Nugroho

E, Maulany RF. Jakarta: Penerbit Buku Kedokteran. 1996. h. 153-64, 177-78, 238-9, 245.

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