NATIONAL UNIVERSITY MICAELA BASTIDAS OF

APURÍMAC

FACULTY OF ENGINEERING

ACADEMIC PROFESSIONAL SCHOOL OF AGROINDUSTRIAL ENGINEERING

Methodology of scientific research

Extraction, physicochemical characterization, and antimicrobial activity of

molle essential oil (schinus molle)

DRA: Liz Bejarano Luján

Nombre CODE:

Lizbeth Cinthia QUISPE RAFAELE 152068

Abancay–Peru

2018
 Extraction, physicochemical characterization and activity

antimicrobial of essential oil of molle (schinus molle)

Figure 1: flowchart of essential oil extraction from leaves ............of

molle (Schinus molle)

Chapter I

Problem statement

The healthcare system currently still faces coverage limitations in

the supply of medications to meet the demands of the national population.

In Peru, various institutions have paid attention to the development of products

natural with varied orientations regarding raw materials, uses, and location of the

industrial plants; among which are the essential oils that have a

growing demand in the national market and can provide income both to the

farmers like producers because currently people are oriented towards

their consumption of alternative products thanks to the fact that they are healthier.

The healthcare needs of the population in general and in particular the

located in urban and rural areas of Peru, are exacerbated by the availability of

medication in these places is insufficient.

Rural populations, due to their ancestral legacy, have practiced healing for their ailments.

resorting to various phytotherapeutic methods, the problem lies in that if these practices

subsists, especially the use of the pepper tree (Schinus molle), in the treatment of wounds, due to the

healing properties of this plant.

In Apurímac, the molle is a tree that grows and is abundant throughout the areas. It grows by

general on the banks of roads, rivers, ravines, etc. Being very well known by the locals

and to which, to date, no technological treatment has been given.

The molle has been identified with secondary metabolites that have insecticidal activity or

antibacterial, obtained from the fruits and leaves of the tree. It contains essential oils

capable of repelling and killing insects (Rodriguez, 2005).

 One of the raw materials with the highest essential oil content is the fruit of the Paraguayan pepper tree.

(Schinus molle), this characteristic motivates the present study, in addition, the molle is a plant

originating from Peru, which has many properties for pharmaceutical use, perfumery, food,

etc.

Essential oils have medicinal properties and have been used since the

antiquity to cure diseases. They are the natural fragrant compounds that occur in

the plants that are isolated from them; generally liquids (on some occasions

semisolid and very rarely solid; poorly soluble in water, but can be volatile with

vapors evaporate at different speeds under atmospheric pressure. Modern science them

process to obtain drugs or specific remedies to prevent or cure various conditions

both of humans and animals. Based on their properties, they are widely

used for the digestive, respiratory, nervous, and circulatory systems. The high demand for

I use natural essential oils in the cosmetic and food industries, especially for essences.

extracted by ecological technology such as steam dragging became a

business alternative. The essential oil of molle is extracted from the fruits of the tree.

Peruvian pink pepper or Andean pepper. It is effectively used for fractures, and in all the

respiratory tract diseases, they also serve as powerful antibiotics and anti-

anti-inflammatory, antibacterial, has proven to be an effective antibacterial medication in

In vitro and having an anti-microbial activity against numerous bacteria and germs

Pathogens in various studies, in addition, the essential oil of molle showed antiviral effect.

against several viruses and showed to be cytotoxic against cancer cells 9kb. (Gonzalez, 2009).

Objective of the research

 General problem

What will be the physicochemical characterization and activity in the yield of

antibacterial properties of molle essential oil (Schinus molle)?

Specific problem

What will be the characterization and yield of essential oil from molle (Schinus

sauce)?

What will be the antibacterial activity of essential oil from molle (Schinus molle)?

General objective

determine the physicochemical characterization and antimicrobial characterization of oil

molle essential (Schinus molle).

1.4 specific objective

Determine the physicochemical characteristics of the essential oil extracted from molle (schinus)

soft)

Identify the characteristics of the essential oil of the molle (Schinus molle).

Evaluate the performance of the essential oil extraction from the fruits of the molle.

(schinus molle).
 Justification of the research

Molle (Schinus molle) is a beautiful tree native to the Andes of Peru.

always green, grows wild and cultivated in dry coastal, mountainous, and jungle areas

up to 3,500 meters above sea level. It grows in light to rocky soils and propagates by seeds.

Tree with an open crown, highly branched and with hanging branches; leaves composed of 15 to

20 leaflets. It has small flowers in panicles and its fruits grow in clusters.

pink, with a seed. The trunk has latex and the leaves of the pepper tree have a smell.

penetrating, characteristic of the species.

The great demand for the use of natural essential oils in the cosmetic industry and

mainly nutritional from essences extracted through ecological technology such as drag

by steam it became a business alternative. The essential oil of molle is extracted

from the fruits of the Peruvian pink pepper tree or Andean molle. It has effective use for

fractures, and in all diseases of the respiratory tract, it also serves as a powerful

antibiotics and anti-inflammatories, antibacterial, has proven to be an effective medication

antibacterial in vitro and have an anti-microbial activity against numerous bacteria and

pathogenic germs in several studies (Bernhard, Shibamoto, Yamaguchi, & White, 1983).

the essential oil of molle demonstrated antiviral effect against several viruses and showed to be

cytotoxic against cancer cells 9kb (Bandoni, 2000).

In addition, essential oils have medicinal properties and have been used since the

antiquity to cure diseases. Modern science processes them to obtain drugs or

specific remedies to prevent or cure various conditions of both humans and

animals. Based on their properties, they are widely used for the digestive system,

respiratory, nervous, and circulatory

They are currently widely used in the world of aromatherapy. These oils provide

to the food industry characteristic flavors and aromas, widely used in bakeries,

jams, candies, soft drinks, ice creams, preservatives, cookies, dairy products, etc. They are used in

the chemical industry to provide fragrances for cleaning products, such as deodorants

environmental, incense, soaps, detergents, dishwashing liquid, hospital use products,

also to insecticides and disinfectants. They are used in the cosmetic industry for the

manufacture of colognes, perfumes, toilet soaps, creams of various types, shampoos,

deodorants, conditioners, and hair sprays, etc. They have various uses in the

agroindustry such as natural insecticides, balanced feed for fattening in pigs and

birds, flavoring in accordance with the processed product, residual plant material, once

extracted oil, used as a feed supplement for cattle, pigs

chicken manure or as natural fertilizer

(Martinez, 2003).

Peru is one of the countries with the greatest biodiversity in the world, many of its species

vegetables can be sustainably utilized by the industry, a clear alternative

it is Schinus molle L., a native tree with medicinal applications, whose essential oil

it exhibits insecticidal, antibacterial, and antifungal properties, demonstrated with extensive

spectrum (Chirino, 2006).

The general objective of this work was to extract and characterize the essential oil from the fruits.

from the molle of the region of Apurímac (provinces of Abancay and Grau)

18.158%

(5,280%) for the fruits of Los Palos; and the moisture content (21.875%), ashes

(3.353%) and proteins (5.560%).

 The fruits of the molle from the provinces of Abancay and Grau have a

similar structure: thin and brittle exocarp (reddish to pink and brown color)

bermejo), resinous mesocarp (thin and cracked beige color), hollow endocarp (little

woody and hard mustard-colored), and a small seed with a very thin testa and embryo

meaty.

Conduct new research on the utilization of essential oil

soft in the food and agro-industrial field, based on this study, since the

the physical-chemical properties of oil and the mixture of its chemical components make it

it is a potentially industrializable product. The fruit can contain 5% oil

essential. Properties and actions: analgesic, antibacterial, antidepressant, antimicrobial,

antifungal, antiviral.

Essential oil extraction diagram of molle

Schinus molle

Raw material

Collection

Heavy

Selection
 Cooling 3 times

4°C
Washing

Crushing

Extraction

Conservation

Ambient cooling

Distillation of oil

Chapter II

Reference framework

1. BACKGROUND

(huamani, 2005) mentions that the steam drag method is one of the oldest,

Most essential oils can be obtained using this method, which consists of

to bring the water where the solid matter to be treated is immersed to a boil and separating the

components by condensation and evaluated the antifungal activity.

In her research work, Development of an Extraction System

Oils, they conducted experiments to obtain greater yields regarding the

essential oil concluding that according to the technique that provides greater advantages, it

select the steam drag extraction method, relate the effectiveness

antimicrobial of the essential oil of Schinus molle L. against Chlorhexidine Gluconate

0.12% from 24 to 72 hours of exposure.

Werner (2005) mentions that there are factors that affect the performance of oils.

essential. Among the factors that directly influence production performance

The following raw essential oils can be listed:

T
 ype of raw material: refers to the genetic characteristics of the molle fruit.

(Schinus molle), since there is still a difference even within the different families; furthermore the

raw material is also influenced by the place and time of production, by the aging

or age of the plant, of the fruits and by the cleanliness in the cutting, plant diseases, etc.

D
 rying time: depending on this drying time, the plant will have more or

less water quantity, which affects the yield.

P
 article size: the transfer area and the number of compartments

open depends on that factor, as well as the steam flow in drag methods with

vapor.

E
 xtraction time: refers to the time of the extraction process, in which the

essential oil from the fruit of molle (schinus molle L.) gradually extracted.

 haracteristics of the extraction equipment: size of the equipment, material of

C

construction.

C
 haracteristics of the flows: it refers to the amount of raw material used, the

volume and the purity of the solvent used, the amount of water or vapor used, its

temperature and pressure.

The extraction method used was steam drag.

 The sample sizes were 15, 30, and 45 grams and the moisture content as

fresh and dry at 10% humidity. The extraction time was two hours and the size of

the particle was approximately 2 mm, as this increases the surface of

contact helping to break the glands that contain the essential oil with

greater ease.

Theoretical framework

2.1 ESSENTIAL OIL OF MOLLIE

The Schinus molle L., commonly called molle (Spanish), mulli (Quechua), cullash,

belongs to the Anacardiaceae family; genus Schinus which is a family of trees from 6 to

8 m in height, with rough bark, with compound leaves of 7 to 25 pairs of leaflets, sessile, of

ash green to light green on both sides. When squeezed, they emit a smell.

characteristic. Its flowers are small, yellowish-white in color. The fruits are drupes.

coral rounded to red-purple when ripe. The seeds are round,

wrinkled when dry, brown to black in color. It has one seed per fruit, It is

a species widely distributed in the mountains of South America and Central America

where it is probably native, it is cultivated in tropical lands and in subtropical areas

throughout the world as an ornamental plant (2). In Peru, it is found more frequently

between 100 and 3,200 meters above sea level on the western slope, as well as in the southern zone of the valley

Mantaro.

In our country, it grows wild and unexploited in the Junín region.

It has a series of pharmacological properties. It is known as molle, aguaribay, pepper.

black, terebinth (among others). It is cultivated for ornamental purposes in gardens and places.

public, due to its somewhat spicy flavor. The leaves to combat swelling of the feet.

In the leaves, bark, and fruits, the essential oil is present, it is a rich source of

triterpenes, sesquiterpenes, and monoterpenes, many of the biological activities are

attributed to the essential oils found in the plant, the essential oil of the pepper tree,

demonstrated antiviral effect against several viruses and showed to be cytotoxic against cancer cells

9kb, and its benefits that have an astringent, purifying, diuretic, balsamic effect, such as

colirio, diurético, en enjuagues, pesticida, depurativo, estomacal, tónico, antiviral, sirven

as well as powerful antibiotics and anti-inflammatory agents for the skin and muscles.

It has dyeing and medicinal properties, known to the ancient Peruvians, of the

boiling its leaves and bark yields a yellow dye that allows for dyeing cotton and

wool. The coloring component is a flavonoid that is still little studied.

2.1.1 The molle

It is an incorruptible tree, with a fragrant smell, and a hot temperament, and with much

stipticity, highly valued by the Indians for its wonderful effects, giving it some

knife cuts on the trunk as on the branches, it exudes a white and fragrant resin, which was used

to embalm and preserve the bodies of the Inca kings without corruption, when they placed them

in their tombs or graves.

The molle is found at altitudes of up to 3,600 meters above sea level, and its distribution range in

South America includes: Ecuador, Paraguay, Peru, southern Brazil, Chile, Uruguay, and Argentina.

The molle plant has a long history of use as an astringent, depurative, and diuretic.

balsamic, as eye drops, diuretic, in rinses, pesticide, purifying, stomachic, tonic,

antiviral.
 Scientific Name Schinus molle

Purity Percentage 95—98

Germination Percentage (%) 50—80

Optimal altitude range 1,000 to 3,200 meters above sea level

Propagation Botanical seed.

Seed viability Media

Seed per average kilogram 30,600

Origin of the Seed Cusco, Junin, La Libertad, Ancash

Family: Anacardiaceae

[Link] Taxonomy of molle (Schinus molle L.)

The classification of the plant under study is shown below (Huamancaja M. y.

1982).

Kingdom: Vegetal

Division: Phanerogam

Class: Dicotyledon

Subclass: Archclamideans

Order: Sapindales

Family: Anacardiaceae

Gender: Schinus

Species: soft

Schinus molle L.

common_name Soft

There are over 70,000 commonly used names to refer to this species.

(Antonio, 2011). In America, some are: 'molle', 'mulli', 'huiñan', 'cullash',

“aguaribay”, “falsa pimienta” (Perú); “aroeira”, “bálsamo sanalotodo” (Colombia);

“bálsamo”, “terebinto”, “aguaribay” (Argentina); “anacahuita”, “aroeira vermelha” (Brasil);

tree of Peru, pirwi, pirú (Mexico); pippertree (United States). Of all of them,

the most widespread are 'aguaribay' and 'molle' (Chicaiza, 2010)

[Link] General description

The molle is a tree several meters high that grows both wild and

grown in dry areas of the coast, highlands, and parts of the Amazon, from sea level

up to 3500 m.a.s.l.

It presents an extended and superficial radical system. Its trunk is gnarled and rough.

reaching a height of 4 to 15m. It has a round and open crown, with petiolated leaves.

and alternately, pointed at the end. Its flowers are very small, whitish in color or

yellowish, and they are arranged in a large number in hanging terminal panicles and

axillary. Their fruits are red, and once dried, they resemble pepper grains. This

the fruit contains a coloring substance and an essential oil, which produces a resin

fragrant, impregnated with turpentine (Bernhard, Shibamoto, Yamaguchi, & White, 1983)

The molle tree is native to South America. 'Molle' derives from the Quechua word 'mulli'.

This tree was noted by many naturalists and travelers of the conquest era.

America. There are references to "molle" trees in the high areas of the Andes and

There are also records of 'molle' or 'aguaribay' in the Las Misiones area (Northeast of

Argentina, Southern Brazil, and Northern Uruguay) (Viturro et al., 2010).

Regarding its origin and common names, the molle is native to the valleys.

interandeans of central Peru. It is found endemically, from southern Mexico

up to northern Chile and central Argentina, especially in Peru (CONABIO, 2010).

According to the writings of Garcilaso de la Vega, the molle was the sacred tree of the Incas.

who planted and watered around their palaces, temples, and public buildings.

Also, a large part of the royal roads they built were shaded by these.
trees still in desert places where the water to water them had to be brought from the hills

through long ditches or channels. From the resin that oozes from the trunk, they made a liquid gum that

It was used for embalming; the mummies owed their state of preservation to the use of this.

antiseptic rubber. In Peru and Bolivia, the fruits were used in those times to prepare

chicha

Form: Evergreen tree 4 m to 8 m (up to 15 m) tall, with a diameter at the height

from the chest of 25 cm to 35 cm.

Crown / Leaves: Rounded and open crown, providing moderate shade. Leaves

composed, alternate from 15 cm to 30 cm long, hanging, with milky sap; odd-pinnate

from 15 to 41 leaflets, generally paired and measuring from 0.85 cm to 5 cm in length, closely

lanceolate, yellowish green color.

Flowers:Axillary panicles on the terminal leaves 10 cm to 15 cm long, very flowers

small and numerous, yellowish in color, measuring 6 mm in width.

Fruits: Drupes in hanging clusters, each fruit 4 mm to 7 mm in diameter, pink or

reddish, with leathery, glossy exocarp, dry at maturity, thin and resinous mesocarp.

Seeds: The seeds have a well-defined embryo that fills the entire cavity;

Endosperm testae are thin, the mesocarp is part of the dispersal unit.

CULTURE: Beautiful tree native to the Andes of Peru and remains always green,

It grows wild and cultivated in dry areas of the coast, mountains, and jungle up to 3,500.

It develops in light to rocky soils and propagates through seeds.

2.2 CHEMICAL COMPOSITION OF ESSENTIAL OIL OF MOLLE

2.2.1 Chemical composition

The photochemical studies of Schinus molle L. have indicated that it contains tannins.

alkaloids, flavonoids, spherical saponins, sterols, terpenes, and essential oil.

(Gonzalez, 2009)
 The phytochemical analysis of the molle reveals that the molle plant contains tannins, alkaloids,

flavonoids, saponins, steroids, sterols, terpenes, gums, resins, and essential oils. The

essential oil is found in the leaves, bark, and fruits, it is a rich source of

triterpenes, sesquiterpenes and monoterpenes, many of the biological activities are

attributed to the essential oils found in the plant.

The fruits can contain up to 5.00% essential oil and the leaves can contain

up to 2.00% essential oil. According to FIGMA Y S.R.L, the yield from seeds

dried is from 5.00%- 7.00 %, being the main component. Some bibliographic sources

it is revealed that the essential oil of molle is made up of chemical compounds

shown below.

3. Conceptual Framework

3.1 DEFINITION OF ESSENTIAL OIL

 They are called the fragrant constituents or 'essences' of a plant. The term

oil, probably, originates from the fact that volatile liquid fractions (substances

responsible for the aroma of the plant) exist in the glands or between the cells in the form

liquid, which like fatty oils are immiscible with water and because it

evaporation by exposure to air at room temperature is called: volatile oils,

essential oils, essential oils or essences.

Essential oils are volatile liquids, mostly insoluble in water, but

easily soluble in alcohol, ether, and vegetable and mineral oils. They are generally not

oily to the touch. For the most part, they have a pleasant smell, although there are some with an unpleasant odor.

relatively unpleasant such as those of the ajoyla onion, which contain

sulfur compounds.

From a chemical point of view, essential oils are a complex mixture of

volatile terpenoids (monoterpenes, sesquiterpenes, diterpenes, etc.) that are produced by

Secondary metabolism of the plant, or the isoprene rule. Initially, oils

essentials were defined as the volatile portion of the plant obtained through distillation

from steam, but volatile oils can also be produced through fractionation of the

oleoresin obtained by solvent extraction (at low or high pressures).

The main compounds that make up an essential oil belong to the

following chemical classes:

Aliphatic compounds, hydrocarbons and their low molecular weight oxygenated analogs

molecular (alkanes, alcohols, aldehydes, ketones, esters, and acids).

Monoterpenes, C10H16

Monoterpenoids, C10H18O, C10H16O, C10H14O.

Sesquiterpenes, C1sH24

Sesquiterpenes, C15H26O, C15H24, C15H22.

 F enilpropanos

Other terpenes like diterpenes (C20H32)and triterpenes (C30that are not

Volatile compounds are widely found in some foods and are bioactive compounds.

very important.

3.2 Applications of essential oils

INDUSTRIAL APPLICATION INDUSTRY

Cosmetics: Preparation of perfumes, toothpaste, creams ointments.

Food Flavorings, scentings.

Liquor store: Flavorings, aromatizers.

Pharmaceutical In veterinary medicine, antiseptics, insect repellents, Effects

positives about central nervous systems.

Fine chemistry: Chemical precursors. Plastics to mask the bad odor that

they have some rubber and plastics.

Hygiene and cleaning: To provide fragrance to cleaning products, air fresheners.

environmental.

3.3 Quality of the essential oil

To obtain good quality of pepper tree oil depends on several factors such as:

Age of the crop: Related to the existence and availability of nutrients of the crop

Weather conditions: Related to brightness, temperature, rainfall,

altitude

Agricultural work: Related to planting distances, fertilizers, pruning, post-harvest.

Washing, drying, packaging, storage, transportation.

Extraction methods: Related to the method used for oil extraction

essential.
 a. Organoleptic characteristics: The characteristics to consider in this are the

color, the color, and the appearance. The first of them is especially important, as many

The uses of essential oils are related to scent (Bandoni, 2000).

[Link] determinations.-It mainly concerns the determination of constants

physical properties, among which density, refractive index, and optical rotation stand out,

miscibility in ethanol, flash point, freezing point. The density can

To be determined with a pycnometer or an electronic densimeter. The temperature must be indicated.

working temperature (usually 20°C). Almost all essences have a density less than that of water.

The refractive index can be determined using an electronic refractometer and is usually measured

also at 20°C, or otherwise a correction is made for temperature difference. This

the parameter is of interest for detecting adulterations. The components of essential oils

they are often optically active, with one optical isomer predominating (Gonzalez,

2009.

3.4 extraction methods, physicochemical characterization and antimicrobial activity

of essential oil of molle (schinus molle)

RAW MATERIAL COLLECTION (FRUITS).-The fruit collection was carried out from the

Víquez locality in the Apurímac region includes: Abancay, Grau, Chincheros.

RECEIPT OF RAW MATERIAL.- In this operation the scale was used.

electrical and the organoleptic maturity of the molle fruit was evaluated.

SELECTION.- The selection was carried out according to the same size, according to the degree of

deterioration or what showed no physical damage was done manually.

 WASHING.- Cleaning was carried out to eliminate the sugars and residues present in the

seed.

DRYING.- Its aim is to remove the water content from the raw material until

to obtain a final humidity of 13%, they were dried at room temperature with intervention of

sun rays, once the seed was dry, the husk that covered it was removed as well as the

leaves and the sticks.

CRUSHING.- The fruit went through a moderate crushing process to reduce the

size of approximately 0.4 to 0.6 cm in order to facilitate the passage of steam and larger

contact area and obtain optimal oil yields; releasing the oil inside,

Being favorable for the extraction, a sieve with a diameter of No. 14 was used.

EXTRACTION. Its aim is to obtain the oil from the raw material.

Aimed for that purpose, the sample was used according to the size of the equipment 1 kg per

process and four stages were considered:

FIRST STAGE OF PRESSURIZATION: The pressure of the gas to be used is raised to

solvent at a value P1 above its critical pressure Pc; this operation is carried out by

medium of a compressor or pump.

SECOND STAGE OF TEMPERATURE ADJUSTMENT: Energy is removed or added.

thermal, with a heat exchanger, or electric resistances, to carry the solvent

compressed to the required extraction temperature, a state that is above its

critical temperature.

THIRD STAGE OF EXTRACTION: The FSC is directed to the extractor where it

find the sample (molle (schinus linnaeus), which contains the solute of interest (solid
 FOURTH STAGE OF SEPARATION: the gas is decompressed to a lower pressure P2

at the critical pressure, the solute is released into a separating vessel, and it is suitable for analysis.

Packaging: Subsequently to the chemical drying, the obtained essential oil was packaged in

amber glass jars.

Stored: It was then stored under refrigeration conditions (4-5 °C), and were

submitted to the corresponding analyses and evaluations.

With this technique, we are able to carry out a process using clean technology and we have the

high extraction effect characteristic, high quality, low temperature extraction,

low energy consumption. And without pollution, especially indicated for extraction of

heat - sensible and active substance.

The greatest advantage of extraction using this method with supercritical fluids is its

less impact on the environment. It is a clean technology because, when using substances

Like CO2, the waste produced is not toxic and is easily recyclable.

possibility of quickly eliminating the excess of supercritical fluid simply

Decompression and collection of the concentrated analytes is another important advantage.