Assessment Of Bioactivity Of Bangladeshi
1325
Advances in Natural and Applied Sciences, 6(8): 1325-1329, 2012
ISSN 1995-0772
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Assessment Of Bioactivity Of Bangladeshi Medicinal Plants Using Brine Shrimp
Lethality Assay
Firoz Ahmed, Fatema Islam, Nusratun Nahar, Mithil Ahmed, Aynal Haque Rana, K.M.
Hasanur Rahman, Md. Mumit Hossain, Mohammed Rahmatullah
Faculty of Life Sciences, University of Development Alternative, Dhanmondi, Dhaka-1205, Bangladesh.
Firoz Ahmed, Fatema Islam, Nusratun Nahar, Mithil Ahmed, Aynal Haque Rana, K.M. Hasanur
Rahman, Md. Mumit Hossain, Mohammed Rahmatullah: Assessment Of Bioactivity Of Bangladeshi
Medicinal Plants Using Brine Shrimp Lethality Assay
ABSTRACT
Twenty six Bangladeshi medicinal plants used in traditional medicines were evaluated for brine shrimp
lethality toxicity. Different solvent extracts of Trachyspermum ammi, Cissampelos pareira, Vetiveria
zizanioides, Cassia angustifolia, Woodfordia fruticosa, Cinnamomum tamala, Neolomarckia cadamba,
Amaranthus viridis, Amaranthus tricolor, Brassica juncea, Brassica oleracea, Raphanus sativus, Curcuma
longa, Curcuma zedoaria, Elettaria cardamomum, Ficus religiosa, Ficus benghalensis, Prunus cerasoides,
Chenopodium album, Spinacia oleracea, Symplocos racemosa, Terminalia chebula, Tinospora cordifolia,
Cyperus rotundus, Pterocarpus santalinus, and Lagenaria siceraria were used in the study. Of the 26 plants
tested, 20 plants (76.9%) were toxic to brine shrimp (LC50 < 30 microg/ml). Among the extracts screened, the
ethanolic extract of Spinacia oleracea leaves and methanolic extract of Amaranthus viridis whole plants had the
highest toxicity to brine shrimp (LC50 = 0.06 microg/ml). The drug vincristine sulfate was considered as
reference standard.
Key words: Cytotoxicity, brine shrimp, Bangladesh, medicinal plants
Introduction
A large percentage of the population of developing countries depends on traditional medicines for their
primary health-care needs (FAO, 2004). Bangladesh has a rich heritage of herbal medicines among the South
Asian countries. The poor and ethnic peoples of Bangladesh rely on medicinal plants as prescribed by traditional
medicinal practitioners for treatment against various diseases. Although a large number of medicinal plants are
used in the traditional medicinal system of Bangladesh, the majority of these plants have not yet undergone
chemical, pharmacological and toxicological studies to investigate their bioactivity (Ghani, 2003). Traditional
records and ecological diversity indicate that Bangladeshi plants represent an exciting resource for possible lead
compounds in drug design and development (Uddin et al., 2011).
Twenty six Bangladeshi medicinal plants (Trachyspermum ammi, Cissampelos pareira, Vetiveria
zizanioides, Cassia angustifolia, Woodfordia fruticosa, Cinnamomum tamala, Neolomarckia cadamba,
Amaranthus viridis, Amaranthus tricolor, Brassica juncea, Brassica oleracea, Raphanus sativus, Curcuma
longa, Curcuma zedoaria, Elettaria cardamomum, Ficus religiosa, Ficus benghalensis, Prunus cerasoides,
Chenopodium album, Spinacia oleracea, Symplocos racemosa, Terminalia chebula, Tinospora cordifolia,
Cyperus rotundus, Pterocarpus santalinus, and Lagenaria siceraria) were collected from various regions of
Bangladesh following accounts of their medicinal uses (Ghani, 2003; Yusuf et al., 1994) or when our ongoing
ethnomedicinal surveys (Nawaz et al., 2009; Rahmatullah et al., 2009a-c; Chowdhury et al., 2010; Hasan et al.,
2010; Hossan et al., 2010; Mollik et al., 2010a,b; Rahmatullah et al., 2010a-g; Haque et al., 2011; Islam et al.,
2011; Jahan et al., 2011; Rahmatullah et al., 2011a,b; Sarker et al., 2011; Das et al., 2012; Rahmatullah et al.,
2012a-d) indicated that they were used by traditional medicinal practitioners for treatment of various ailments.
Extracts of various plant parts were screened for their cytotoxic activities.
Cytotoxic potential of whole plant or plant part extracts were measured by the brine shrimp (Artemia
salina) nauplii lethality assays. This assay is a useful tool for isolation of bioactive compounds from plant
extracts (Sam, 1993). The assay was first proposed by Michael et al. (1956), and later developed by Vanhaecke
Corresponding Author: Dr. Mohammed Rahmatullah Pro-Vice Chancellor University of Development Alternative House
No. 78, Road No. 11A (new) Dhanmondi R/A, Dhaka-1205 Bangladesh
E-mail: rahamatm@hotmail.com; Fax: 88-02-8157339
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Adv. in Nat. Appl. Sci., 6(8): 1325-1329, 2012
et al. (1981) and Sleet and Brendel (1983). We used this assay for determination of the cytotoxic potential of the
26 Bangladeshi medicinal plants in the present study.
Materials and Methods
Plant materials:
The plant samples were collected from various regions of Bangladesh based on their ethnomedicinal uses.
To determine whether a plant has any ethnomedicinal use, surveys were conducted among folk and tribal
medicinal practitioners as described before in the Introduction. All plants were identified at the Bangladesh
National Herbarium at Dhaka where voucher specimens were also deposited. The various plant parts were cut
into small pieces, air-dried under shade and grounded using a laboratory mill and blender.
Extraction:
A known amount of dried powdered sample (usually 100g) was extracted by maceration with methanol or
chloroform at a ratio of plant material to solvent of 1:5. Collected extracts were then filtered and concentrated in
vacuo at 40oC. The solvent-free extracts were kept refrigerated at 4oC and used for cytotoxicity assays.
Toxicity testing against brine shrimp:
Hatching shrimp. Brine shrimp eggs, Artemia salina leach were hatched in artificial seawater prepared by
dissolving 38g of sea salt in 1L of distilled water. The pH of the solution was adjusted to 8.5. After 48h
incubation at room temperature (26-30oC) under constant aeration, the larvae (nauplii) were attracted to one side
of the vessel with a light source and collected with a pipette. Nauplii were separated from eggs by aliquoting
them three times in small beakers containing seawater.
Brine shrimp assay. The bioactivity of the extracts was monitored by the brine shrimp lethality test (Meyer
et al., 1982). Samples were dissolved in dimethylsulfoxide (DMSO) and diluted with artificial sea salt water so
that final DMSO concentration did not exceed 0.05%. 50 l of 2000 g of the plant extract was placed in one
sample tube and a two-fold dilution carried out down the column of sample tubes. The last sample tube was left
with sea salt water and DMSO only to serve as the drug-free control. The total volume was adjusted to 5 ml with
sea salt water. 100 l of suspension of nauplii containing 10 larvae was added into each tube and incubated for
24h. The tubes were then examined under a magnifying glass and the number of dead nauplii in each tube
counted. Experiments were conducted with control (vehicle treated), and different concentrations of the test
substances in a set of three tubes per dose. Vincristine sulfate was used as a positive control in all experiments.
Statistical analysis:
Lethality assays were evaluated by Finney computer statistical program to determine the LC50 values and
95% confidence intervals. All data were expressed as mean (Microg/ml) (Zhao et al., 1992; McLaughlin, 1991).
Results and Discussion
The cytotoxic activity of different plant parts or whole plants of 26 Bangladeshi traditionally used
medicinal plants were investigated against brine shrimp (Artemia salina) in vitro. The results are shown in Table
1. All the crude extracts of different plant species resulting in LC50 values of less than 200 microg/ml were
considered active against brine shrimp. McLaughlin (1991) has reported that the results obtained with Artemia
salina are quantitative and reproducible, and the activities parallel cytotoxicities. As a general observation, ED50
values for cytotoxicities will fall one order of magnitude (10 times) lower than LC50 values for brine shrimp.
According to the standards of the American National Cancer Institute (NCI), ED50 values of ≤ 20 microg/ml for
not pure compounds are considered active (Cordell et al., 1993), so we can take a level for the median lethal
concentration (LC50) as 200 microg/ml. About 80% of the plant parts or whole plants in different solvents used
in the present study exhibited toxic effects to brine shrimp at LC50 values of lower than 30 microg/ml. The
Amaranthaceae and Chenopodiacea family plants exhibited the most cytotoxicity in these brine shrimp lethality
assays. It is interesting that of the two Chenopodiaceae family plants, Chenopodium album reportedly prevented
progression of cell growth and enhanced cell toxicity in human breast cancer cell lines (Khoobchandani et al.,
2009). Possible anti-tumor promoters have also been reported in Spinacia oleracea (Wang et al., 2002). The
results suggest that brine shrimp lethality assays can be a good indicator for locating plants with anti-cancer
activities.
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Adv. in Nat. Appl. Sci., 6(8): 1325-1329, 2012
In the present study, DMSO was used as the solvent and as negative control. This is in accordance with
previous report that brine shrimp nauplii can tolerate up to 11% of DMSO (Sam, 1993). Further studies are
being conducted to isolate and purify the bioactive constituents for further evaluation in human cell line cultures
for cytotoxic effects.
Table 1: The LC50 values of different Bangladeshi medicinal plant extracts against Artemia salina.
Serial
Scientific name of plant
Family
Part extracted
Solvent used for
number
extraction
1
Trachyspermum ammi Apiaceae
(L.) Sprague ex Turrill
2
Cissampelos pareira L.
Menispermaceae
3
Vetiveria zizinioides (L.) Poaceae
Nash.
4
Cassia
angustifolia Fabaceae
Vahl.
5
Woodfordia
fruticosa Myrtaceae
(L.) Kurz.
6
Cinnamomum
tamala Lauraceae
(Ham.) Nees & Eberm.
7
Neolomarckia cadamba Rubiaceae
(Roxb.) Bosser
8
Amaranthus viridis L.
Amaranthaceae
9
Amaranthus tricolor L.
Amaranthaceae
10
Brassica juncea (L.) Cruciferae
Czern.
11
Brassica oleracea L.
Cruciferae
12
Raphanus sativus L.
Cruciferae
13
Curcuma longa L.
Zingiberaceae
14
Curcuma
zedoaria Zingiberaceae
Roscoe
15
Elettaria cardamomum Zingiberaceae
Maton
16
Ficus religiosa L.
Moraceae
17
Ficus benghalensis L.
Moraceae
18
Prunus cerasoides D. Rosaceae
Don.
19
Chenopodium album L.
Chenopodiaceae
20
Spinacia oleracea L.
Chenopodiaceae
21
Symplocos
racemosa Symplocaceae
Roxb.
22
Terminalia
chebula Combretaceae
(Gaertn.) Retz.
23
Tinospora
cordifolia Menispermaceae
(Willd.) Hook. f. &
Thomson
24
Cyperus rotundus L.
Cyperaceae
25
Pterocarpus santalinus Fabaceae
L.
26
Lagenaria
siceraria Cucurbitaceae
(Molina) Standl.
Vincristine
sulfate
(standard)
The results are presented as LC50 values (microg/ml).
Fruit
Ethanol
Probit Analysis (Mean)
Finney
method
[Lognormal distribution]
(microg/ml)
11.11
Root
Root
Ethanol
Ethanol
40.10
67.48
Leaf
Ethanol
109.35
Flower
Ethanol
140.89
Leaf
Ethanol
142.50
Leaf
Methanol
36.72
Whole plant
Whole plant
Leaf
Methanol
Methanol
Ethanol
0.06
0.36
17.04
Leaf
Leaf
Rhizome
Rhizome
Ethanol
Ethanol
Ethanol
Ethanol
7.81
0.13
3.88
0.16
Fruit
Ethanol
18.51
Bark
Bark
Stem
Ethanol
Ethanol
Ethanol
13.84
6.82
0.75
Whole plant
Leaf
Stem
Methanol
Ethanol
Ethanol
0.07
0.06
2.90
Fruit
Ethanol
1.53
Stem
Ethanol
15.29
Root
Stem
Ethanol
Ethanol
1.03
22.03
Leaf
Ethanol
23.26
0.62
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Advances in Natural and Applied Sciences, 6(8): 1325-1329, 2012
ISSN 1995-0772
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Assessment Of Bioactivity Of Bangladeshi Medicinal Plants Using Brine Shrimp
Lethality Assay
Firoz Ahmed, Fatema Islam, Nusratun Nahar, Mithil Ahmed, Aynal Haque Rana, K.M.
Hasanur Rahman, Md. Mumit Hossain, Mohammed Rahmatullah
Faculty of Life Sciences, University of Development Alternative, Dhanmondi, Dhaka-1205, Bangladesh.
Firoz Ahmed, Fatema Islam, Nusratun Nahar, Mithil Ahmed, Aynal Haque Rana, K.M. Hasanur
Rahman, Md. Mumit Hossain, Mohammed Rahmatullah: Assessment Of Bioactivity Of Bangladeshi
Medicinal Plants Using Brine Shrimp Lethality Assay
ABSTRACT
Twenty six Bangladeshi medicinal plants used in traditional medicines were evaluated for brine shrimp
lethality toxicity. Different solvent extracts of Trachyspermum ammi, Cissampelos pareira, Vetiveria
zizanioides, Cassia angustifolia, Woodfordia fruticosa, Cinnamomum tamala, Neolomarckia cadamba,
Amaranthus viridis, Amaranthus tricolor, Brassica juncea, Brassica oleracea, Raphanus sativus, Curcuma
longa, Curcuma zedoaria, Elettaria cardamomum, Ficus religiosa, Ficus benghalensis, Prunus cerasoides,
Chenopodium album, Spinacia oleracea, Symplocos racemosa, Terminalia chebula, Tinospora cordifolia,
Cyperus rotundus, Pterocarpus santalinus, and Lagenaria siceraria were used in the study. Of the 26 plants
tested, 20 plants (76.9%) were toxic to brine shrimp (LC50 < 30 microg/ml). Among the extracts screened, the
ethanolic extract of Spinacia oleracea leaves and methanolic extract of Amaranthus viridis whole plants had the
highest toxicity to brine shrimp (LC50 = 0.06 microg/ml). The drug vincristine sulfate was considered as
reference standard.
Key words: Cytotoxicity, brine shrimp, Bangladesh, medicinal plants
Introduction
A large percentage of the population of developing countries depends on traditional medicines for their
primary health-care needs (FAO, 2004). Bangladesh has a rich heritage of herbal medicines among the South
Asian countries. The poor and ethnic peoples of Bangladesh rely on medicinal plants as prescribed by traditional
medicinal practitioners for treatment against various diseases. Although a large number of medicinal plants are
used in the traditional medicinal system of Bangladesh, the majority of these plants have not yet undergone
chemical, pharmacological and toxicological studies to investigate their bioactivity (Ghani, 2003). Traditional
records and ecological diversity indicate that Bangladeshi plants represent an exciting resource for possible lead
compounds in drug design and development (Uddin et al., 2011).
Twenty six Bangladeshi medicinal plants (Trachyspermum ammi, Cissampelos pareira, Vetiveria
zizanioides, Cassia angustifolia, Woodfordia fruticosa, Cinnamomum tamala, Neolomarckia cadamba,
Amaranthus viridis, Amaranthus tricolor, Brassica juncea, Brassica oleracea, Raphanus sativus, Curcuma
longa, Curcuma zedoaria, Elettaria cardamomum, Ficus religiosa, Ficus benghalensis, Prunus cerasoides,
Chenopodium album, Spinacia oleracea, Symplocos racemosa, Terminalia chebula, Tinospora cordifolia,
Cyperus rotundus, Pterocarpus santalinus, and Lagenaria siceraria) were collected from various regions of
Bangladesh following accounts of their medicinal uses (Ghani, 2003; Yusuf et al., 1994) or when our ongoing
ethnomedicinal surveys (Nawaz et al., 2009; Rahmatullah et al., 2009a-c; Chowdhury et al., 2010; Hasan et al.,
2010; Hossan et al., 2010; Mollik et al., 2010a,b; Rahmatullah et al., 2010a-g; Haque et al., 2011; Islam et al.,
2011; Jahan et al., 2011; Rahmatullah et al., 2011a,b; Sarker et al., 2011; Das et al., 2012; Rahmatullah et al.,
2012a-d) indicated that they were used by traditional medicinal practitioners for treatment of various ailments.
Extracts of various plant parts were screened for their cytotoxic activities.
Cytotoxic potential of whole plant or plant part extracts were measured by the brine shrimp (Artemia
salina) nauplii lethality assays. This assay is a useful tool for isolation of bioactive compounds from plant
extracts (Sam, 1993). The assay was first proposed by Michael et al. (1956), and later developed by Vanhaecke
Corresponding Author: Dr. Mohammed Rahmatullah Pro-Vice Chancellor University of Development Alternative House
No. 78, Road No. 11A (new) Dhanmondi R/A, Dhaka-1205 Bangladesh
E-mail: rahamatm@hotmail.com; Fax: 88-02-8157339
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Adv. in Nat. Appl. Sci., 6(8): 1325-1329, 2012
et al. (1981) and Sleet and Brendel (1983). We used this assay for determination of the cytotoxic potential of the
26 Bangladeshi medicinal plants in the present study.
Materials and Methods
Plant materials:
The plant samples were collected from various regions of Bangladesh based on their ethnomedicinal uses.
To determine whether a plant has any ethnomedicinal use, surveys were conducted among folk and tribal
medicinal practitioners as described before in the Introduction. All plants were identified at the Bangladesh
National Herbarium at Dhaka where voucher specimens were also deposited. The various plant parts were cut
into small pieces, air-dried under shade and grounded using a laboratory mill and blender.
Extraction:
A known amount of dried powdered sample (usually 100g) was extracted by maceration with methanol or
chloroform at a ratio of plant material to solvent of 1:5. Collected extracts were then filtered and concentrated in
vacuo at 40oC. The solvent-free extracts were kept refrigerated at 4oC and used for cytotoxicity assays.
Toxicity testing against brine shrimp:
Hatching shrimp. Brine shrimp eggs, Artemia salina leach were hatched in artificial seawater prepared by
dissolving 38g of sea salt in 1L of distilled water. The pH of the solution was adjusted to 8.5. After 48h
incubation at room temperature (26-30oC) under constant aeration, the larvae (nauplii) were attracted to one side
of the vessel with a light source and collected with a pipette. Nauplii were separated from eggs by aliquoting
them three times in small beakers containing seawater.
Brine shrimp assay. The bioactivity of the extracts was monitored by the brine shrimp lethality test (Meyer
et al., 1982). Samples were dissolved in dimethylsulfoxide (DMSO) and diluted with artificial sea salt water so
that final DMSO concentration did not exceed 0.05%. 50 l of 2000 g of the plant extract was placed in one
sample tube and a two-fold dilution carried out down the column of sample tubes. The last sample tube was left
with sea salt water and DMSO only to serve as the drug-free control. The total volume was adjusted to 5 ml with
sea salt water. 100 l of suspension of nauplii containing 10 larvae was added into each tube and incubated for
24h. The tubes were then examined under a magnifying glass and the number of dead nauplii in each tube
counted. Experiments were conducted with control (vehicle treated), and different concentrations of the test
substances in a set of three tubes per dose. Vincristine sulfate was used as a positive control in all experiments.
Statistical analysis:
Lethality assays were evaluated by Finney computer statistical program to determine the LC50 values and
95% confidence intervals. All data were expressed as mean (Microg/ml) (Zhao et al., 1992; McLaughlin, 1991).
Results and Discussion
The cytotoxic activity of different plant parts or whole plants of 26 Bangladeshi traditionally used
medicinal plants were investigated against brine shrimp (Artemia salina) in vitro. The results are shown in Table
1. All the crude extracts of different plant species resulting in LC50 values of less than 200 microg/ml were
considered active against brine shrimp. McLaughlin (1991) has reported that the results obtained with Artemia
salina are quantitative and reproducible, and the activities parallel cytotoxicities. As a general observation, ED50
values for cytotoxicities will fall one order of magnitude (10 times) lower than LC50 values for brine shrimp.
According to the standards of the American National Cancer Institute (NCI), ED50 values of ≤ 20 microg/ml for
not pure compounds are considered active (Cordell et al., 1993), so we can take a level for the median lethal
concentration (LC50) as 200 microg/ml. About 80% of the plant parts or whole plants in different solvents used
in the present study exhibited toxic effects to brine shrimp at LC50 values of lower than 30 microg/ml. The
Amaranthaceae and Chenopodiacea family plants exhibited the most cytotoxicity in these brine shrimp lethality
assays. It is interesting that of the two Chenopodiaceae family plants, Chenopodium album reportedly prevented
progression of cell growth and enhanced cell toxicity in human breast cancer cell lines (Khoobchandani et al.,
2009). Possible anti-tumor promoters have also been reported in Spinacia oleracea (Wang et al., 2002). The
results suggest that brine shrimp lethality assays can be a good indicator for locating plants with anti-cancer
activities.
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Adv. in Nat. Appl. Sci., 6(8): 1325-1329, 2012
In the present study, DMSO was used as the solvent and as negative control. This is in accordance with
previous report that brine shrimp nauplii can tolerate up to 11% of DMSO (Sam, 1993). Further studies are
being conducted to isolate and purify the bioactive constituents for further evaluation in human cell line cultures
for cytotoxic effects.
Table 1: The LC50 values of different Bangladeshi medicinal plant extracts against Artemia salina.
Serial
Scientific name of plant
Family
Part extracted
Solvent used for
number
extraction
1
Trachyspermum ammi Apiaceae
(L.) Sprague ex Turrill
2
Cissampelos pareira L.
Menispermaceae
3
Vetiveria zizinioides (L.) Poaceae
Nash.
4
Cassia
angustifolia Fabaceae
Vahl.
5
Woodfordia
fruticosa Myrtaceae
(L.) Kurz.
6
Cinnamomum
tamala Lauraceae
(Ham.) Nees & Eberm.
7
Neolomarckia cadamba Rubiaceae
(Roxb.) Bosser
8
Amaranthus viridis L.
Amaranthaceae
9
Amaranthus tricolor L.
Amaranthaceae
10
Brassica juncea (L.) Cruciferae
Czern.
11
Brassica oleracea L.
Cruciferae
12
Raphanus sativus L.
Cruciferae
13
Curcuma longa L.
Zingiberaceae
14
Curcuma
zedoaria Zingiberaceae
Roscoe
15
Elettaria cardamomum Zingiberaceae
Maton
16
Ficus religiosa L.
Moraceae
17
Ficus benghalensis L.
Moraceae
18
Prunus cerasoides D. Rosaceae
Don.
19
Chenopodium album L.
Chenopodiaceae
20
Spinacia oleracea L.
Chenopodiaceae
21
Symplocos
racemosa Symplocaceae
Roxb.
22
Terminalia
chebula Combretaceae
(Gaertn.) Retz.
23
Tinospora
cordifolia Menispermaceae
(Willd.) Hook. f. &
Thomson
24
Cyperus rotundus L.
Cyperaceae
25
Pterocarpus santalinus Fabaceae
L.
26
Lagenaria
siceraria Cucurbitaceae
(Molina) Standl.
Vincristine
sulfate
(standard)
The results are presented as LC50 values (microg/ml).
Fruit
Ethanol
Probit Analysis (Mean)
Finney
method
[Lognormal distribution]
(microg/ml)
11.11
Root
Root
Ethanol
Ethanol
40.10
67.48
Leaf
Ethanol
109.35
Flower
Ethanol
140.89
Leaf
Ethanol
142.50
Leaf
Methanol
36.72
Whole plant
Whole plant
Leaf
Methanol
Methanol
Ethanol
0.06
0.36
17.04
Leaf
Leaf
Rhizome
Rhizome
Ethanol
Ethanol
Ethanol
Ethanol
7.81
0.13
3.88
0.16
Fruit
Ethanol
18.51
Bark
Bark
Stem
Ethanol
Ethanol
Ethanol
13.84
6.82
0.75
Whole plant
Leaf
Stem
Methanol
Ethanol
Ethanol
0.07
0.06
2.90
Fruit
Ethanol
1.53
Stem
Ethanol
15.29
Root
Stem
Ethanol
Ethanol
1.03
22.03
Leaf
Ethanol
23.26
0.62
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