CET 96

DOI: 10.3303/CET2296057

Paper Received: 15 February 2022; Revised: 23 June 2022; Accepted: 4 July 2022
Please cite this article as: Sandoval A.N., Mendoza K.L., Lloclla H., Villanueva L.N., Rojas J.C., 2022, Lethality of Hydroalcoholic Extracts from
Fruit Plant Leaves in the Peruvian Jungle, Chemical Engineering Transactions, 96, 337-342 DOI:10.3303/CET2296057

CHEMICAL ENGINEERING TRANSACTIONS

VOL. 96, 2022

A publication of

The Italian Association
of Chemical Engineering
Online at www.cetjournal.it

Guest Editors: David Bogle, Flavio Manenti, Piero Salatino
Copyright © 2022, AIDIC Servizi S.r.l.

ISBN 978-88-95608-95-2; ISSN 2283-9216

Lethality of Hydroalcoholic Extracts from Fruit Plant Leaves in
the Peruvian Jungle

Ana N. Sandoval*a, Karla L. Mendozaa, Herry Llocllaa, Ludwig N. Villanuevaa, Jose
C. Rojasb
a

Universidad César Vallejo, Perù
bUniversidad Nacional de San Martín, Perù
asandoval@ucv.edu.pe

Lethality of hydroalcoholic extract from fruit plant leaves in the Peruvian jungle in Artemia salina was evaluated.
The type of study was experimental with a control group (K2Cr2O7). The biological material was fruit leaves
(Cocos nucifera, Mauritia flexuosa, Theobroma cacao L, Coffea sp, and Musa sp) collected in San Martín
Region; Likewise, the phytochemical march to the leaves was carried out to identify their active principles. The
Artemia salina eggs were provided by the department of animal physiology of the National University of Trujillo,
they were kept under specific conditions such as artificial light, a temperature of 25 ° C and a time of 24 hours,
allowing them to mature up to 48 hours. For the preparation of the hydroalcoholic extract it was by the maceration
method using 500g of leaves and 500mL of 70° alcohol; for 15 days under stirring, the solution was taken to a
vertical rotary evaporator to obtain a dry extract preparing concentrations of 10, 100, 250, 500 and 1000 μg/mL.
The sample consisted of 10 larvae for each plant species and concentration, performing the test in triplicate.
The LC50 lethality of Artemia salina in the samples was classified as: ˃1000 μg/mL. (Non- lethality), 500 ˂
LC50 ≤ 1000 (Low toxicity), 100 ˂ LC50 ≤ 500 (Moderate lethality), LC50 ˂ 100 (High lethality). It was obtained
as a result that Mauritia flexuosa and Theobroma cacao L. in concentrations 10, 100 μg/mL present high and
moderate lethality.

1. Introduction

Medicinal plants have been used since ancient times as a curative means, receiving interest for their healing
potential due to their active components (Afsar et al., 2015). Nowadays, the consumption of medicines has
raised their costs which has generated limited access for the population, opting for the use of medicinal plants
for the treatment of diseases as part of primary health care (WHO, 2015; Teles and Costa, 2014). The use of
botanical and plant-derived medicines was valued at $23.2 and $24.4 million between 2013 and 2014 and is
expected to reach $25.6 million in 2015 and $35.4 billion in 2020 (BCC, 2017). The ethnobotanical use of plants
is important because it allows research and through it the discovery of new therapeutic alternatives. Studies
have shown that plant parts such as seeds, fruits, leaves and roots have been used for disease control (Moraia
et al., 2019). However, a phytochemical analysis must be performed to identify the bioactivity possessed by
each active compound to avoid side effects (Ullah et al., 2014).
The Peruvian rainforest has a great diversity of flora including fruit plants of species Cocos nucifera, Mauritia
flexuosa, Theobroma cacao L., Coffea sp and Musa sp (MINAM, 2019); species that are used as an alternative
for traditional medicine due to their active principles, such as steroids, phenolic compounds, flavonoids,
terpenes, reducing sugars, lactones, among others (Pereira et al., 2016; Sandoval et al., 2020). Some research
has shown that hydroalcoholic extracts of leaves of some species are toxic for human consumption due to the
combination of their active compounds (Leite et al, 2015; Paredes et al., 2018; Sandoval et al., 2020). Artemia
salina is a light brown shrimp of the Crustaceae family, with a size of 1 to 7 mm. It is cosmopolitan, lives in salt
water at a temperature of 6ºC to 35ºC, and feeds on algae and bacteria. The study of its physiology concludes
in performing preliminary tests because they are low cost, easy to handle and present minimum requirements
for laboratory manipulation. Toxicology studies indicate the presence of sensitivity to certain toxic agents and

337

therefore provide reliable results. As this is a practical, sustainable and sustainable method, it is used to evaluate
the pharmacological potential of synthetic and natural compounds measured through their lethality in plants,
which implies only life or death (Avalos et al., 2014; Silva et al., 2015). Therefore, the objective of the study was
to evaluate the lethality of hydroalcoholic extracts of Peruvian rainforest fruit leaves on Artemia salina.

2. Method

2.1. Vegetal material

500 grams of leaves of the species Cocos nucifera, Mauritia flexuosa, Theobroma cacao L., Coffea sp and Musa
sp, were collected in the district of Cacatachi, San Martín, at 295 m. above sea level and 12 km north of Tarapoto
(6°29'40" south latitude and 76°27'57" west longitude). The samples were placed in vacuum bags and labeled
with their name at a temperature of 37 °C. Afterwards, they were taken to the Truxillense Herbarium of the
Universidad Nacional de Trujillo (National University of Trujillo) for identification and deposit with a registration
code for each species: Cocos nucifera (COD. 59603), Mauritia flexuosa (COD.59597), Theobroma cacao L.
(COD. 59599), Coffea sp (COD. 59609) and Musa sp (59608).

2.2 Preparation of hydroalcoholic extract

The leaves were washed with distilled water and disinfected with 70° ethanol. They were fractioned to an
approximate size of 4 mm. For the extraction of the hydroalcoholic extract, the maceration method was used:
350 g of leaves and 500 mL with 70° ethanol for 15 days under agitation with a vertical rotary evaporator
(Scilogex RE-100) at 75 revolutions per minute to obtain dry extracts. Dilutions at concentrations of 10, 100,
250, 500 and 1000 μg/mL were prepared with the sample obtained.

3.3 Phytochemical analysis

The hydroalcoholic extract of fruit leaves was evaluated in order to identify its active principles. Each sample
was subjected to solvents of increasing polarity to obtain secondary metabolites according to their solubility
using reagents and dyes to determine the presence or absence of active components such as: steroids,
triterpenes, quinones, phenolic compounds, flavonoids, lactones, alkaloids, reducing sugars, tannins and
saponins, by using the protocol described by Lock (2016). 3 mL of pure extract was added to 10 test tubes to
identify secondary metabolites through color change, classified as light, moderate or strong. The tests used to
determine the presence of each type of secondary metabolite are listed in Table 1.

Table1. Phytochemical analysis of the hydroalcoholic extract of the leaves of medicinal plants from the

Peruvian Jungle.

Assay Secondary metabolites
Cocos

nucífera

Coffea

Theobroma

cacao L

Musa

Mauritia

flexuosa

Lieberman-Bouchard Steroids and triterpenes (++) (+) (++) (++) (++)
Borntrager Quinones (-) (-) (-) (-) (-)

Ferric chloride Phenolic compounds (+) (+) (+) (++) (+++)

Shinoda Flavonoids (+) (+) (-) (+++) (+++)

Baljet Lactones (+) (-) (-) (++) (+)

Dragendorff Alkaloids (-) (+) (-) (-) (-)
Mayer Alkaloids (-) (+) (-) (-) (-)
Fehling Sugar reducers (++) (+) (++) (++) (++)
Gelatin Tannins (++) (+) (-) (-) (++)
Foam Saponins (-) (-) (-) (-) (-)

Note: Color changes of secondary metabolites in (+) = slight, (++) = moderate and (+++) = strong.

2.4 Obtaining and breeding Artemia salina

The 20-day-old Artemia salina eggs were provided by the Department of Animal Physiology, Universidad
Nacional de Trujillo, and were washed with filtered seawater to remove impurities. An incubation chamber with
abundant oxygenation was used, adding to this container one gram of eggs (equivalent to 700-800 eggs),
allowing incubation in 5 liters of filtered seawater under artificial fluorescent light at 110 Watts, temperature of
25 °C and adjusted to a pH of 7-8 for 24 h. The Artemia salina eggs were fed with commercial yeast extract to
hatch and continue their biological cycle for approximately seven days. Then, 10 7-day-old larvae per each

338

concentration (10, 100, 250, 500 and 1000 μg/mL) at stage III were used as a toxicity marker due to its high
sensitivity (Silva et al., 2015; Jaramillo et al., 2016).

2.5 Lethality testing

Concentrations of 10, 100, 250, 500 and 1000 µg/mL of filtered seawater were prepared according to the
protocol described by Seremet et al (2018). Then 5 µg of extract was diluted in 5 mL of filtered seawater,
equivalent to 10 μg/mL, 50 μg of extract in 5 mL of filtered seawater, equivalent to 100 μg/mL, 125 μg of extract
in 5 mL of filtered seawater, equivalent to 250 μg/mL, 250 μg of extract in 5 mL of filtered seawater, equivalent
to 500 μg/mL and 500 μg of extract in 5 mL of filtered seawater, equivalent to 1000 µg/mL.Ten larvae were
placed in a test tube containing 10 mL of filtered seawater and 0.5 mL of the hydroalcoholic extract; this was for
each plant species and concentration. Each assay was performed in triplicate and a K2Cr2O7 control group of
250 μg/mL was used for comparison (Goncalves et al., 2019; Simoes and Almeida, 2015). The larvae were
exposed to the treatments for 24 hours; after this time the number of dead larvae was counted only if there was
no movement of their appendages for 10 seconds (Socea et al., 2015); for this purpose, a stereoscope (Eurolab
NSD-405) was used.
The toxicity criteria for Artemia salina samples were classified as follows: ˃ 1000 µg/mL (non-toxic), 500 LD 50
≤1000 (low toxicity), 100 ˂ LD 50 ≤ 500 (moderate toxicity), LD 50 ˂ 100 (high toxicity) (Alonso et al., 2017 and
Monteiro et al., 2018). The toxicity percentage of the organisms exposed to the effect of the extract was
estimated as follows:

Toxicity (percentage) = (TNA - NAA) / TNA*100 (1)
Where: TNA = Total number of Artemia salina.
NAA = Number of live Artemia salina (Jan and Khan, 2016).

2.6 Ethical Statement

Artemia salina does not represent a danger to the environment. It is not an endangered species, as it does not
appear on the red list of the International Union for Conservation of Nature (IUCN), the species is used for
scientific purposes (IUCN, 2019).

3. Results

It is observed that the 5 plant species have high lethality compared to the positive control group, with Mauritia
flexuosa having the highest percentage of lethality.

Figure 1. Concentration of 10μg/mL of hydroalcoholic extract vs. positive control group (K2Cr2O7).

73
80 83

86 90

55 55 55 55 55

100

Coffea sp Cocos
nucífera

Musa sp Theobroma
cacao L

Mauritia
flexuosa

C
a
s
e
f

a
ta

li
ty

r
a
te

Concentration of hydroalcoholic extract μg/mL

10µg/ml Control + (K2Cr2O7)

339

Figure 2. Concentration of 100μg/mL of hydroalcoholic extract vs. positive control group (K2Cr2O7).

It is evident that the 4 plant species have high lethality compared to the positive control group, with Mauritia
flexuosa having the highest percentage of lethality.

Figure 3. Concentration of 250μg/mL of hydroalcoholic extract vs. positive control group (K2Cr2O7).

It is observed that the 4 plant species have moderate lethality (Coffea sp, Cocos nucífera, Musa sp and Mauritia
flexuosa) compared to the positive control group, with Mauritia flexuosa having the highest percentage of
lethality.

Figure 4. Concentration of 500μg/mL of hydroalcoholic extract vs. positive control group (K2Cr2O7).

63 66
73

55 55 55 55 55

100

Coffea sp Cocos
nucífera

Musa sp Theobroma
cacao L

Mauritia
flexuosa

C
a
s
e
f

a
ta

li
ty

r
a
te

Concentration of hydroalcoholic extract μg/mL

100µg/ml Control + (K2Cr2O7)

56
53

60
55 55 55 55 55

100

Coffea sp Cocos
nucífera

Musa sp Theobroma
cacao L

Mauritia
flexuosa

C
a
s
e
f

a
ta

li
ty

r
a
te

Concentration of hydroalcoholic extract μg/mL

250µg/ml Control + (K2Cr2O7)

48
43

44
55 55 55 55 55

100

Coffea sp Cocos nucífera Musa sp Theobroma
cacao L

Mauritia
flexuosa

C
a
s
e
f

a
ta

li
ty

r
a
te

Hydroalcoholic extract concentration μg/mL

500µg/ml Control + (K2Cr2O7)

340

It is evident that the 5 plant species have low lethality compared to the positive control group, which has the
highest percentage of lethality.

Figure 5. Concentration of 1000μg/mL of hydroalcoholic extract vs. positive control group (K2Cr2O7).

It is observed that the 5 plant species do not present lethality compared to the positive control group, which
presents the highest percentage of lethality.

4. Conclusions

The active compounds of the hydroalcoholic extracts of fruit leaves from the Peruvian rainforest were analyzed,
finding steroids, triterpenes, phenolic compounds, flavoniods and tannins. Artemia salina was used as an
indicator to measure lethality. The results indicated that concentrations less than or equal to 100 μg/mL have
high lethality, concentrations of 250 μg/mL moderate lethality, concentrations of 500 μg/mL low lethality and
concentrations equal to or greater than 1000 μg/mL are not lethal. The hydroalcoholic extracts with the highest
lethality concentration were Mauritia flexuosa and Theobroma cacao L. The consumption of medicinal plants
has been increasing due to their probable effectiveness. However, indiscriminate use is a latent risk due to the
toxicity of some compounds within the plant that can cause collateral damage. For this reason, it may be useful
to study medicinal plant extracts to show therapeutic or toxic activity of their active compounds. Other studies
indicate that if a sample is not lethal to Artemia salina, its effects will also be similar for humans.

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149sandoval.pdf
Lethality of Hydroalcoholic Extracts from Fruit Plant Leaves in the Peruvian Jungle
aUniversidad César Vallejo, Colombia
bUniversidad Nacional de San Martín, Colombia