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Biologically Active Natural Products from Indonesian Medicinal Plants

This e-mail address is being protected from spambots. You need JavaScript enabled to view it ,1)* Tati Herlina,1) Euis Julaeha,1) Dikdik Kurnia,1) Tri Mayanti,1) Desi Harneti,1)
Nurlelasari,1) Rani Maharani,1) Tomoyuki Fujita2) and Hideo Hayashi3)

1)Department of Chemistry, Faculty of Mathematics and Natural Sciences,
Universitas Padjadjaran, Jatinangor 45363, Indonesia

2)Sciences of Functional Foods (Integrated Department), Graduate School of Agriculture,
Shinshu University 3304 Minami-minowa, Kami-ina, Nagano, 399-4598 Japan

3)Division of Life Sciences, Graduate School of Life and Enviromental Sciences,
Osaka Prefecture University, Sakai, Osaka 599-8531 Japan


Abstract

During the course of investigation of Indonesian medicinal plants, several bioactive principles have been isolated and chemically identified, such as insecticidal bufadienolides from Indonesian Kalanchoe plants (1), paralytic erythrina alkaloids from Indonesia Eryhtrina plants (2),  anti-malarial triterpenoid from Erythrina plants (3), anti-malarial flavonoid from Eryhtrina plants (4), anti-fertility steroid from Clerodendron serratum (4), anti-feedant triterpenoid from Lansium domesticum (5), cytotoxic triterpenoid from Toona sureni and Toona sinensis (Meliaceae) (6) and anti-tumor triterpenoid from Aglai smithi (7). The structures of those active compounds were identified on the basis of spectroscopic evidence and comparison of those related data previously reported. This paper presents some recent progress in those chemical investigations.


Introduction

In course of our continuing search for novel bioactive substances from indonesian medicinal plants, we started the scientific exploration in 1997 “investigation of naturally occurring drug materials in Indonesia” which comprises two subjects: (1) the survey of traditional folk medicine in Indonesia and (2) screening of bioactive compound such i) insecticidal and paralytic against silkworm (Bombyx mori), ii) anti-malarial against Plasmodium falcifarum, iii) anti-fertility against spermatozoa Rectus domesticus, iv) anti-feedant against Epilachna sparsa and v) cytotoxicity against Artemia salina. The exploration has been carried out in collaboration with Laboratory of Plant Taxonomy, Department of Biology, Universitas Padjadjaran, Indonesia and financially supported by the Directorate General of Higher Education, Ministry of National Education, Indonesia. We have so far collected 160 specimens in the fields, and since 1997 we have undertaking the chemical analysis of their bioactive principles. This paper presents some recent progress in those chemical investigations.


Results and Discussions

I. Insecticidal Compounds

Kalanchoe (Crassulaceae)

During the course of our continuing search for novel insecticidal compounds, the methanolic extract of Kalanchoe plants showed insecticidal activity against silkworm bioassay.1) A Kalanchoe plant, known as “sosor bebek” in Indonesia is a perennial herb and has succulent leaves. The plant is known in folklore and traditional medicine in Indonesia for the treatment of fever, abscesses, bruises, contused wounds, coughs and skin diseases.2,3)

a) Kalanchoe pinnata

Leaves of K. Pinnata were collected from plantation trees growing in Bandung District, West Java, Indonesia in August 1997. The methanolic extract of the fresh leaves of K. pinnata was concentrated and extracted with dichloromethane. The dichloromethane extract exhibited an insecticidal activity toward silkworms. The dichloromethane extract was partitioned between n-hexane and methanol containing 10% water. The active lower layer was extracted with ethyl acetate. By using the insecticidal activity to follow the separations, the ethyl acetate fraction was separated by combination of column chromatography on Wakaogel C-200, Kieselgel 60, and preparative HPLC on RP-18 to afford a new insecticidal bufadienolide, bryophyllin C (1), and a known bufadienolide, bryophyllin A (2), together with an inactive bersaldegenin-3-acetate (3).4)


b)    Kalanchoe daigremontiana x tubiflora

Leaves of K. daigremontiana x tubiflora were collected from plantation trees growing in Bandung District, West Java, Indonesia in July 1999. The methanolic extract of the fresh leaves of K. pinnata was concentrated and extracted with dichloromethane. The dichloromethane extract exhibited an insecticidal activity toward silkworms. The dichloromethane extract was partitioned between n-hexane and methanol containing 10% water. The active lower layer was extracted with ethyl acetate. By using the insecticidal activity to follow the separations, the ethyl acetate fraction was separated by combination of column chromatography on Wakagel C-200, Kieselgel 60, and preparative TLC on Kieselgel GF254 to afford a new insecticidal bufadienolide, methyl daigremonate (4) and three known bufadienolides, bersaldegenin-1,3,5-orthoacetate (5) daigremontianin (6), and bersaldegenin-1-acetate (7).5)

 

c).  Kalanchoe daigremontiana

Leaves of K. daigremontiana were collected from plantation trees growing in Bandung District, West Java, Indonesia in August 2000. The methanolic extract of the fresh leaves of K. daigremontiana was worked up as described for the leaves of K. pinnata and K. daigremontiana x tubiflora to yield two known insecticidal bufadienolides, bersaldegenin-1,3,5-orthoacetate (5) and daigremontianin (6).

 

d).   Structure-Insecticidal Activity Relatioships of Bufadienolides

In order to confirm the functional groups responsible for insecticidal activity, bufadienolides 2 and 6 were chemically transformed by treating with a base, followed by treating with an acid according to the partially modified procedure of a previous report6,7) to yield methyl isobryophyllinate A (8) and methyl isobersaldegenin-1,3,5-orthoacetate (9).

The insecticidal activity of bufadienolides (1-7) together with two synthetic derivatives (8 and 9) were evaluated against the third instars larvae of silkworm according to a procedure described previously.1) Insecticidal activity of these compounds are summarized in Table 1 using LD50 (50% lethal dose) values. Of these bufadienolides, bufadienolides having both orthoacetate and a-pyrone opening type of bufadienolides like in 3, 4, 7, 8, and 9 showed a weak or no activity at concentration of 100 mg/g of diet. These results indicated that an orthoacetate and a-pyrone moieties were essential structural elements for exhibiting the insecticidal activity.

In addition, daigremontianin (6) having oxygenated substituents at C-11 and C-12 showed the strongest activity among these compounds, indicating that the oxygenated substituents at C-11 and C-12 in ring C enhanced the insecticidal activity.8)

Table.1.  Insecticidal Activity of Bufadienolidesa)

Compounds LD50
(mg/g of diet)
Bryophyllin C (1) 3
Bryophyllin A (2) 5
Bersaldegenin-3-acetate (3) > 100
Methyl daigremonate (4) 0.9
Bersaldegenin-1,3,5-orthoacetate (5) 20
Daigremontianin (6) 80
Bersaldegenin-1-acetate (7) > 100
Methyl isobryophyllinate (8) > 100
Methyl isobersaldegenin-1,3,5-orthoacetate (9) > 100

a) Oral administration

 

e)    Anti-tumor Promoting Activity of Bufadienolides from Kalanchoe plant

Five bufadienolides (1, 2, 3, 5 and 6) isolated from the leaves of Kalanchoe plants were examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells induced by tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All bufadienolides showed inhibitory activity, and bryophyllin A (2) exhibited the most marked inhibition (IC50 = 0.4 mM) among the tested compounds. Bryophyllin C (1), a reduction analogue of 1, and bersaldegenin-1,3,5-orthoacetate (3) lacking the orthoacetate moeity were less active. These results strongly suggest that bufadienolides are potential cancer chemopreventive agents.9)

 

I. Paralytic Compounds

Erythrina (Leguminosae)

During the course of our continuing search for novel insecticidal compounds, the methanolic extract of Erythrina plants showed paralytic activity against silkworm bioassay.1) An Erythrina plant, known as “dadap” in Indonesia has been used as a folk medicine for treatment of fever, child-birth, and eye diseases in Indonesia.10)

a)        Erythrina subumbrans (Leguminosae)

Samples of the leaves of E. subumbrans were collected in October, 2001, in Bandung District, West Java, Indonesia. The plant was identified by a staff at the Laboratory of Plant Taxonomy, Department of Biology, Bandung Institute of Technology, Bandung, Indonesia, and a voucher specimen has been deposited at the herbarium. The methanolic extract of the dried leaves of E. subumbrans was concentrated and extracted with CH2Cl2. The CH2Cl2 extract exhibited a paralytic activity toward silkworms. The CH2Cl2 extract was partitioned between n-hexane and methanol containing 10% water. The active lower layer was extracted with ethyl acetate. By using the paralytic activity to follow the separations, the ethyl acetate fraction was separated by combination of column chromatography on Wakogel C-200, Kieselgel 60 and preparative TLC on ODS KC 18F to afford four erythrina alkaloids, erythratidinone (10), 3-demethoxyerythratidinone (11), erysotrine (12) and erythratidine (13).11)

In the further search for novel paralytic alkaloid from E. subumbrans, the methanolic extract of the bark of E. Subumbrans showed significant paralytic activity against silkworm. The methanolic extract of the dried bark of E. subumbrans was worked up as described for the dried leaves previously and yield two paralytic erythrina alkaloids, 7-en-erythratidine (14) and 3,15,16-trimethoxy-2,8-dioxoerythrinane (15).12)

 

a) Erythrina fusca

In the further screening for paralytic activity from Indonesian Erythrina plants, the methanolic extract of the seed of E. fusca exhibited the significant paralytic activity against the third instar larvae of silkworm. Samples of the seeds of E. fusca were collected in July, 2002, in Bandung District, West Java, Indonesia. In the previous investigation known that alkaloid compound showed paralytic activity so that further investigation to search alkaloid. The methanolic extract of the seed of E. fusca was acidified with 1% hydrochloric acid to pH 2-3 and then partitioned with dichloromethane to yield an active aquoeous exctract. The aquoeous exctract was bacidified with ammonium hydroxide to pH 9-10 and then extracted with dichloromethane to afford an active dichloromethane. By using the alkaloid test to follow the separations, the dichloromethane extract furthermore was separated by combination of column chromatography on Wakagel C-200 and Kieselgel 60 to yield erythrina alkaloids, 15,16-demethoxy erysothrin (16), erythravin (17) and 1,2,3,4-tetrasubstituted erythrina alkaloid (18).13)

 

c.    Erythrina poeppigiana

Samples of the bark of E. poeppigiana were collected in August, 2003, in Subang District, West Java, Indonesia. The methanolic extract of the dried bark of E. poeppigiana was worked up as described for the seed of E. fusca previously and yield two paralytic erythrina alkaloids, 3-hydroxy-16,17-dimethoxy-8,20-dioxoerythrinan (19) and erythratidine (13).14)

d).   Structure-Paralytic Activity Relatioships of Erythrina Alkaloids

The paralytic activity of erythrina alkaloids (10-19) were evaluated against the third instars larvae of silkworm according to a procedure described previously.6,7) Paralytic activity of these compounds are summarized in Table 2 using ED50 (50% efective dose) values). Of these erythrina alkaloids, alkaloids having oxygenated substitute showed most active indicated that oxygenated substitute was essential structural elements for exhibiting the paralytic activity.

Table.2.  Paralytic Activity of Erythrina Alkaloidsa)

Compounds LD50
(mg/g of diet)
Erythratidinone (10)
15
3-demethoxyerythratidinone (11)
26
Erysotrine (12)
9
Erythratidine (13)
7
7-en-erythratidine (14)
70
3,15,16-trimethoxy-2,8-dioxoerythrinane (15)
44
15,16-demethoxy erysothrin (16)
43
Erythravin (17)
37
1,2,3,4-tetrasubstituted erythrina alkaloid (18)
28
3-hydroxy-16,17-dimethoxy-8,20-dioxoerythrinan (19) > 100

a) Oral administration


III. Anti-malarial Compounds

During the course of our continuing search for novel biologically active natural products from Indonesian medicinal plants, the methanolic extract of the bark of Erythrina variegata (Legimunosae) showed a remarkable anti-malarial activity agaisnt Plasmodium falciparum FCR-3/A and 3D7 strain (resistance to chloroquine) and K1 (sensitive to chloroquine).

Erythrina Variegata (Leguminosae)

The dried leaves of E. variegata was extracted with methanol exhaustively to yield concentrated methanol extract. Furthermore, the methanol extract was extracted succesively with n-hexane, ethyl acetate and n-butanol and then each fraction was evaluated their anti-malarialactivity against P. falcifarum strain 3D7 and K1(Tabel 3).15)

Table.3.  Anti-malarial activity of ethyl acetate and n-butanol extracta)

Samples LD50
(mg/g of diet)
3D7 K1
Methanol extract
> 60 x 103
6.8 x 103
Ethyl acetate extract
17 x 103
27 x 103
n-butanol extract
13 x 103
5.1 x 103
chloroquine
40
40
Artemesinine) 10
10

 

The ethyl acetate extract was separated by combination of column chromatography on Wakogel C-200, Kieselgel G 60 and ODS to yield three active compounds, pentacyclic triterpenoid, 3,22,23-trihydroxy-oleane-12-ene (20), pentacyclic triterpenoid, 3b,11a-28trihydroxy-oleane-12-ene (21) and 10,11-dioxoerythratidine (22).

The n-butanol extract was worked up as described for the ethyl acetate extract to yield an isoflavon derivative, warangalone [8(3,3-dimethyl-alyl)-4’hydroxy-2’’’,2’’’,2’’’-dimethylpyran (23).16)

Compounds 20-23 were evaluated their anti-malarial activity against P. falciparum by in vitro test as showed in Tabel 4. Compound 1 showed IC50 value higher than the others compound against P. falcifarum FCR-3/A strain resistence to chloroquine, whereas compound 2 showed IC50 value higher than the other compounds against P. falcifarum 3D7 strain sensitive to chloroquine.17)

Table.4.  Anti-malarial activity of compounds 21-23

Compounds
IC50 (mg/mL)
FCR-3/A
K1
3D7
Compound 20
0.243
- -
Compound 21
- 23.5
4.3
Compound 22
- 3.3
25.5
Compound 23
-
2.5
4.1

Compounds 22 and 23 showed IC50 value higher than compound 21 against P. falcifarum K1 strains resistence to chloroquine. This results indicated that compounds 20-23 have a remarkable anti-malarial activity but lower than chloroquine and artemisinine. Compounds 20 and 21 as a new triterpenoid pentacyclic derivative that found in Erythrina genera and have anti-malarial activity, whreas compound 23 as an isoflavonoid derivative that found previously has anti-inflamation activity18) and reported to be the first time has anti-malarial in this study.18) Compound 23 showed anti-malarial higher than compounds 21 and 22 due to the presence of epoxy group in pyran ring.

 

IV. Anti-fertility Compounds

In the further of our continuing search for novel biologically active substance from Indonesian medicinal plants, we found that the methanolic extract of the leaves of Clerodendron serratum (Verbenaceae) showed a significance anti-fertlity activity against spermatozoa of white male rat (Rattus novergicus) by in vitro test.

 

Clerodendron serratum (Verbenaceae)

C. serratum (Verbenaceae) is a tropical medicinal plant, original from Asia. It is distributed from Pakistan and India east-ward to Central China and Southern China, Thailand, Malaysia, and Indonesia. In Java, the leaves are used for mouthwash, rheumatism, painful, and consumed during labour19). In the C. serratum plant is known containing flavonoids: luteolin, luteolin 7-O-b-D-glucuronide, scutellarein;  phenolic: (±)-catechin; steroids: a-spinasterol, stigmasterol; triterpenoids:  queretaroic acid,  seratagenic acid20,21), and saponin22). In the leaves contain potassium, a little sodium, alkaloid, and flavonoid flavon. In the root bark there are glicoside phenol, mannitol, and sitosterol25). The ethanol extract of C. serratum leaves has been reported to possess pest-control antifertility26). So far, there is no information about chemical structure of an antifertility compound from C. serratum.

Samples of the leaves of C. serratum were collected in April 2003, in Sumedang District, West Java Province. The plant was identified by a staff at the Herbarium Bandungiensis, Department of Biology, Bandung Institute of Technology, Bandung, and a  voucher specimen has been deposited at herbarium.

The dried C. serratum leaves (4 kg) were soaked in  petroleum ether, and then in methanol to give 493 g of residue. The methanol residue was partitioned between 2% tartaric acid solution  and EtOAc. The EtOAc fraction  (20 g) was chromatographed on Kieselgel G60 using n-hexane : EtOAc as eluent with increasing 10% polarity started to give 8 fraction. The third fraction  was chromatographed on Kieselgel G60  eluted with n-hexane : EtOAc : Me2CO (7:1:1) to give 8 fraction. The second  fraction was repeated purified by the same adsorben eluted with n-hexane: Me2CO (8:1), to give 11 fraction. The third fraction L323 was recrystallized in EtOAc to give C30 sterol compound (24).23)

 

V. Anti-feedant Compounds

In the further of our continuing search for novel biologically active substance from Indonesian medicinal plants, we found that the methanolic extract of the seed of Lansium domesticum Corr (Meliaceae) showed a significance anti-feedant activity against the fourth instars larvae of Epilachna sparsa.

 

Lansium domesticum Corr (Meliaceae)

L. domesticum Corr, a higher and endemic plant of Indonesia. It growns on tropical regions in South East Asia, India and Brazil, is one of the most important traditional medicine used for the treatment anti-malarial.24)

Lansium domesticum Corr. (Meliaceae) was collected in Cililin, Bandung, Indonesia, in 2006. The plant was identified by the staff at Department of Biology, Padjadjaran University.

The dried and milled seeds of L. domesticum was extracted exhaustively by methanol at room temperature. The methanol extract (84 g) was partitioned between n-hexane and 10% aqueous methanol to give an n-hexane soluble fraction (4 g). The n-hexane extract were subjected to column chromatography on silica gel 60 by using step gradient of n-hexane and dichloromethane (8:2). The fraction eluted by n-hexane/dichloromethane (6:4) was further separated by column chromatography on silica gel (n-hexane:ethyl acetate 7:3) to give a new triterpenoid pentacyclic, kokosanolide (25),25) 4-deoxo-kokosanolide (26), onoceradienedione (27), a,g-onoceradiendione (28), and 14-hidroksi-7-onoceradiendione (29).

The mode of action of anti-feedant activity was studied by observe the physiological change of the third instar larvae of Epilachna sparsa which was added by isolated compound on 5% concentration. The test solution of isolated compound was made on 5% solution and added on surface of the leaves of Leunca and put in petri dish. Four instar larvae of Epilachna sparsa was put in petri dish and the another petri dish with four instar larvae of E. sparsa with leaves was added with methanol as a control.  Evaluation was carried out after 24 hours after treatment and then all of the larvae was fixated for preparation of preparate. Compounds 25-29 were evaluated their anti-feedant activity against the third instar larvae of E. sparsa as showed in Tabel 4.

Table 5.  Anti-feedant activity of compounds 25-29

Compounds % Activity
Compound 25
78
Compound 26
0
Compound 27
99
Compound 28
56
Compound 29 85

Compound 27, showed the most active among isolated compound due to the symetrical skelatone whereas compound 26 showed the least active due to polarity.

 

VI.Cytotoxic Compounds

During the course of our continuing search for novel biologically active substance from Indonesian Meliaceae plants, we found that the methanolic extract of the bark of Toona sureni showed a significance cytotoxic activity against Brine Shrimp Lethality Test (BSLT).

 

Toona sureni (Meliaceae)

T. sureni is a plant belong to Meliaceae family and distributed in Nepal, India, Bhutan, Myanmar, Indo-China, Southern China, Thailand and Malaysia peninsula. In Indonesia     T. sureni distributed in Sumatera, Java, and Sulawesi. A part of this plant, especially bark and root were used for treatment diare. Leaves have anti-biotic effect whereas stembark and fruit were used for essential oil.26)

The stem barks of T. sureni were collected from Bogor Botanical Garden in West Java Indonesia and determinated at Herbarium Bogoriense Bogor West Java. Toxicity of compound 1 and 2 was determined by brine shrimp lethality assay (BSLT). Bioindicator brine shrimps nauplii (Artemia salina) hatched at Organic Laboratory Department of Chemistry Unpad.

The dried bark of T. sureni (4 kg) was extracted succesively with n-hexane, ethyl acetate and methanol. The ethyl acetate crude extract was fractionated by vacuum liquid chromatographed on Silica gel G60 eluted by n-hexane, ethyl acetate and methanol step wise to give eleven fraction. The elevent fraction were combined based on their thin layer chromatography (TLC) pattern into five fraction (1-5). Fraction 2 was fractionated by VLC (Silica gel G60) and eluted by n-hexane-ethyl acetate with increasing polarity 10% to give eleven fraction (2A-2K). Fraction 2I (163.6 mg) was subjected to chromatographed on Silica G60 (230-400 mesh) and eluted with chloroform-ethyl acetate (8:2). Fraction 5-7 which eluted  with chloroform-ethyl acetate (8:2) were combined based on their TLC pattern to give 28.1 mg mixture of compound (fraction 2I5). Fraction 2I5 was subjected to ODS chromatography coloum and eluted by step gradient of water-methanol with decreasing polarity 1% (water-methanol (1:9) to obtain triterpenoids, 30 (12.5 mg) and 31 (6.2 mg). 27)

Compounds 30 and 31 showed cytotoxicity against the BSLT at dose of LC50 13.4 and 47.1 ppm, respectively, indicated the sugar moiety decrease cytotoxicity activity.

 

 

Toona sinensis (Meliaceae)

T. sinensis is one of the Meliaceae’s family showed potent cytotoxic activity and has been used traditionally  for the treatment of fever, detoxify, eye infection, and enteristis.28)

The stem bark of T. sinensis was extracted exhaustively with n-hexane, ethyl acetate and methanol. The ethyl acetate extract showed most active against BSLT was further separated using chromatographic methods to afford active compounds 32 and 33. The chemical of active compounds were determined on the basis of spectroscopic methods and comparison with those related data reported previously and identified as catechin and bicatechin, respectively.29)

Compounds 32 and 33 showed cytotoxicity against the BSLT at dose of LC50 30.3 and 36.4 ppm, respectively.

 

Aglaia smithii (Meliaceae)

During the course of our continuing search for cytotoxic compound from Indonesian Meliaceae plant, the methanol extract of Aglaia smithii showed a potent Cytotoxic activity against BSLT.  A. smithii is one of the Aglaia species of the Meliaceae family which is potential producer of cytotoxic  compounds.30)

The stem bark of A. smithii was extracted exhaustively with n-hexane, ethyl acetate and methanol. The ethyl acetate extract showed most active against BSLT was further separated using chromatographic methods to afford active compounds 34 and 35. The chemical of active compounds were determined on the basis of spectroscopic methods and comparison with those related data reported previously and identified as a stigmasterol (34) and aglinine A (35).31)

Compounds 34 and 35 showed cytotoxicity against the BSLT at dose of LC50 58.3, and 17.1 ppm, respectively, indicated the change of ring A increase cytotoxicity activity.

 

 

Acknowledgments

The authors express their sincere gratitude to Dr. Kazuhiro Irie of the Division of Applied Life Sciences in the Graduate School of Agriculture at Kyoto University for the MS measurements, Dr. Kohki Akiyama Division of Life Sciences, Graduate School of Life and Enviromental Sciences, Osaka Prefecture University for NMR and MS measurements, Dr. Safrudin of the Eijkman Institute for anti-malarial activity, Drs. Cucu Hidayat of the Department of Biology, Padjadjaran University for anti-fertility activity. They are also grateful to Mr. Djuandi at Laboratory of Plant Taxonomy, Department of Biology, Institut Teknologi Bandung, Bandung, Indonesia, for collection and identification of the plant materials. They are indebted to Drs. Joko Kusmoro at Laboratory of Plant Taxonomy, Department of Biology, Padjadjaran University, jatinangor, Indonesia, for collection and identification of the plant materials.

 

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