CHAPTER ONE

INTRODUCTION

1.1: Background of the Study

Phytochemical constituents are biologically active compounds found in plants. These

compounds play an important role in the human diet, as they have been shown to have

various health benefits, including antioxidant, anticancer, and anti-inflammatory properties

(Balasundram et al., 2006). As a result, phytochemical constituents have become a topic of

interest in the fields of plant science, nutrition, medicine, and agriculture. The extraction of

phytochemical constituents from plants is a crucial step in the isolation and characterization

of these compounds. Over the years, various methods have been developed and utilized to

extract phytochemicals from different parts of plants, including leaves, stems, flowers, fruits,

and roots. The choice of extraction method depends on several factors, including the type of

plant material, the phytochemical of interest, and the intended use of the extract (Yin et al.,

2019).

Many communities worldwide have been using medicinal plants in their healthcare systems

from time immemorial. As far as Africa is concerned, medicinal plants are still a principal

component of the traditional healthcare system and may be the earliest and the most robust of

all curative entities (Mahomoodally, 2013). In most of rural Africa, traditional practitioners'

prescriptions of plant remedies are the most readily available and affordable medicinal drugs

accessible to the community and, every so often, the only treatments available. Studies have

been performed worldwide to establish the efficacy of plant remedies, and some of the

promising potential results have consequently led to the synthesis of plant-based medicines

(Sofowora et al., 2013). It is estimated that globally, the market value for all medicinal plant

commodities transcends USD 100 billion per year (Vasisht et al., 2016). In present times,

despite the phenomenal growth in the development of synthetic drugs in pharmaceutical

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chemistry, almost 75 to 80% of the global population use herbal drugs as medicines, mostly

in third world countries, for primary health care because of their better tolerability with the

human body and minor side effects, and also easier availability (Vasisht et al., 2016). It has

been documented those natural products are used to develop an estimated 44% of all novel

drugs, primarily as lead compounds, to develop and prepare partially synthetic medicines

(Zhang et al., 2020). Moreover, there has been a paradigm shift to using plants to discover

novel lead molecules and drugs. Research on finding novel drugs in medicinal plants involves

screening the plant extracts for new compounds and then conducting biological activity tests.

Suspected new molecules or bioactive compounds are then isolated and purified for

molecular structure elucidation and further pharmacological or toxicological tests (Kebede et

al., 2021). The research typically involves the following steps: Firstly, plant materials are

harvested from their natural environments. Secondly, using well-preserved leaves, flowers, or

fruits, the plant species is identified by a botanist at herbarium. Thirdly, plant parts of interest

such as roots, stems, backs, leaves, or fruits are treated with an appropriate solvent to extract

the phytochemicals. The extract is then concentrated by processes that aim to remove the

solvent. Then, the extract is subjected to chromatographic techniques to isolate and purify the

bioactive compounds. Further, the isolated compounds may be subjected to various

spectroscopic analyses such as UV/Vis, IR, carbon and proton NMR, and mass spectrometry

for structure determination. After that, chemical methods may be used for pharmacological

and toxicological testing. Finally, the elucidated bioactive compounds may be synthesized or

semi synthesized. Extraction is an essential feature in natural product research. There is

relentless stride going on to improve and discover better extractive techniques having better

efficiency and cost-effectiveness. A wide range of conventional and modern extraction

techniques, their optimization conditions, and their comparative advantages and

disadvantages are discussed in detail. A vast array of recent applications of these techniques

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have also been critically analysed. This literature analysis will be beneficial for advancing the

current and discovering novel extraction techniques.

The aim of this work is to provide a comprehensive overview of the methods used for the

extraction of phytochemicals from plants. The focus will be on the various techniques used

for extraction, including traditional methods such as maceration and percolation, as well as

modern methods such as supercritical fluid extraction, microwave-assisted extraction, and

ultrasound-assisted extraction.

1.2 Definition of Phytochemical constituents

Phytochemical constituents are biologically active compounds found in plants. These

compounds have various functions in plants, including protecting them from pests and

diseases, and attracting pollinators. In recent years, phytochemical constituents have become

a topic of interest in the fields of nutrition, medicine, and agriculture, as they have been

shown to have various health benefits for humans. Phytochemical constituents are found in

various parts of plants, including leaves, stems, flowers, fruits, and roots. They are

responsible for the colour, taste, and smell of plants, and play a crucial role in the plant's

survival. The term "phytochemical" is derived from the Greek words "phyto," meaning plant,

and "chemical," meaning compound (Kris-Etherton et al., 2002).

There are thousands of different types of phytochemicals, and they can be broadly

categorized into several groups based on their chemical structure (Hanhineva et al., 2010).

Some of the major groups of phytochemicals include polyphenols, carotenoids, terpenes, and

alkaloids.

Polyphenols are the largest group of phytochemicals and are found in many plant-based

foods, such as fruits, vegetables, and tea. They are known for their antioxidant properties,

which can protect against damage caused by free radicals in the body (Fiedor et al., 2014).

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Some of the common polyphenols include flavonoids, phenolic acids, and lignans.

Carotenoids are another group of phytochemicals that are responsible for the yellow, orange,

and red colours in many fruits and vegetables. They are also known for their antioxidant

properties and have been shown to have various health benefits, including reducing the risk

of certain types of cancer and eye diseases. Terpenes are a large group of phytochemicals that

are responsible for the distinctive smells and flavours of many plants. They are also used in

the production of essential oils, which have various therapeutic properties (Gonzalez-Burgos

et al., 2012). Alkaloids are a group of phytochemicals that contain nitrogen atoms and are

found in many plants, including coffee, tea, and cocoa. They have various pharmacological

properties and have been used for centuries in traditional medicine to treat a wide range of

ailments.

The health benefits of phytochemicals have been extensively studied over the years, and

research has shown that they can help prevent and treat various diseases, including cancer,

cardiovascular disease, and neurodegenerative diseases (Ninfali et al., 2015). Some of the

mechanisms by which phytochemicals exert their beneficial effects include reducing

inflammation, modulating the immune system, and inhibiting the growth of cancer cells.

However, it is important to note that the health benefits of phytochemical constituents are

largely dependent on the amount consumed, the source of the phytochemical constituents,

and the form in which they are consumed. For example, some phytochemicals are more

bioavailable when consumed with certain types of fats, while others are more bioavailable

when consumed with certain types of carbohydrates. In addition, the processing and cooking

of plant-based foods can also affect the levels of phytochemicals. For example, boiling or

microwaving vegetables can reduce the levels of certain phytochemicals, while steaming or

stir-frying can help preserve their levels. However, it is important to consume phytochemicals

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in the right amounts and from the right sources, and to consider the effects of processing and

cooking on their levels.

1.2.1 Benefits of Phytochemical constituents

Phytochemical constituents are naturally occurring compounds found in plants that have been

studied for their health benefits. They are not considered essential nutrients, but they can have

a positive impact on human health (Biesalski, 2017). Phytochemical constituents have been

shown to have anti-inflammatory properties. Chronic inflammation has been linked to various

chronic diseases, including heart disease, cancer, and Alzheimer’s disease. Studies have

shown that certain phytochemicals, such as curcumin found in turmeric, can reduce

inflammation in the body (Grosso et al., 2017). Curcumin has been found to reduce markers

of inflammation, such as C-reactive protein (CRP) and interleukin-6 (IL-6) in the blood

(Rondanelli et al., 2012).

Phytochemicals can also have antioxidant properties. Antioxidants protect the body from

damage caused by free radicals, which are molecules that can cause oxidative stress and lead

to cellular damage. Many phytochemicals, such as carotenoids found in fruits and vegetables,

have antioxidant properties. Studies have shown that a diet rich in fruits and vegetables can

reduce oxidative stress and improve overall health. Phytochemicals may also have anti-cancer

properties. Studies have shown that certain phytochemicals, such as those found in

cruciferous vegetables like broccoli and cauliflower, can help prevent cancer. These

phytochemical constituents have been shown to reduce the risk of several types of cancer,

including breast, lung, and colon cancer. Other phytochemical constituents, such as those

found in green tea, have also been found to have anti-cancer properties. Phytochemical

constituents may have a positive impact on cardiovascular health. Certain phytochemical

constituents, such as those found in red wine and dark chocolate, have been found to improve

cardiovascular health. These phytochemical constituents have been shown to reduce blood

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pressure and improve blood flow. They may also reduce the risk of heart disease by reducing

the levels of LDL cholesterol in the blood.

Phytochemical constituents may also have a positive impact on brain health. Certain

phytochemicals, such as those found in blueberries, have been shown to improve cognitive

function (Chen et al., 2009). These phytochemical constituents have been found to improve

memory and reduce the risk of age-related cognitive decline. Other phytochemicals, such as

those found in turmeric, have been found to have neuroprotective properties. In addition to

these health benefits, phytochemicals can also help with weight management. Many

phytochemical constituents, such as those found in green tea and chili peppers, have been

found to have thermogenic properties. These phytochemical constituents can increase

metabolic rate, which can help with weight loss.

It is important to note that the benefits of phytochemical constituents are not isolated to

individual compounds. Whole foods and dietary patterns may have greater impact on health

outcomes than individual compounds. For example, a diet rich in fruits, vegetables, whole

grains and lean protein has been shown to have numerous health benefits. While further

research is needed to fully understand the health benefits of phytochemical constituents,

current research suggests that they can have a positive impact on human health.

1.2.2 Sources of Phytochemical constituents

Phytochemical constituents are classified based on their chemical structure and can be found

in a wide range of plant-based foods, including fruits, vegetables, whole grains, legumes, and

nuts. Fruits are a rich source of phytochemical constituents, including flavonoids,

carotenoids, and phenolic acids. Citrus fruits such as oranges and grapefruits contain

flavonoids, particularly hesperidin and naringenin, which have been linked to reduced risk of

cardiovascular disease and improved insulin sensitivity. Berries such as strawberries,

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blueberries, and blackberries are rich in anthocyanins, which are potent antioxidants that

protect against cellular damage caused by oxidative stress. Pomegranates contain ellagic acid,

a polyphenol that has been linked to anti-cancer properties (Higdon et al., 2007).

Vegetables are another excellent source of phytochemical constituents. Cruciferous

vegetables such as broccoli, cauliflower, and cabbage contain glucosinolates, which have

been linked to anti-cancer properties. Green leafy vegetables such as spinach and kale are

rich in carotenoids, particularly lutein and zeaxanthin, which protect against age-related

macular degeneration. Tomatoes are a good source of lycopene, a carotenoid that has been

linked to reduced risk of prostate cancer. Whole grains, including wheat, barley, and oats,

contain various phytochemical constituents, including lignans and phenolic acids. Lignans are

plant compounds that have been linked to reduced risk of breast cancer, while phenolic acids

are antioxidants that protect against cellular damage caused by oxidative stress (Li et al.,

2019). Legumes, including beans, lentils, and chickpeas, are an excellent source of

phytochemical constituents, including flavonoids and lignans. Soybeans contain isoflavones,

a class of phytoestrogens that have been linked to reduced risk of breast cancer and improved

bone health. Nuts and seeds are rich sources of phytochemical constituents, including lignans,

flavonoids, and phenolic acids. Flaxseeds contain lignans, which have been linked to reduced

risk of breast cancer. Walnuts are rich in polyphenols, particularly ellagitannins, which have

been linked to reduced risk of cardiovascular disease.

It is important to note that the content and distribution of phytochemicals in plant-based foods

can vary significantly based on several factors, including variety, ripeness, season, and

processing. For example, the phytochemical content in fruits and vegetables can be affected

by factors such as soil quality, temperature, and sunlight exposure. Additionally, processing

methods such as cooking and freezing can affect the bioavailability of phytochemicals.

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Therefore, it is essential to consume a varied diet rich in whole plant-based foods to ensure

optimal intake of these important compounds.

1.2.3 Bioactive Compounds

A plant’s life produces two categories of vast phytochemicals (Rehab et al., 2018). In the first

category are the primary metabolites. These are required for a plant’s normal growth and

development. They include nucleic acids, carbohydrates, fatty acids, proteins, and the

molecules present in all plants for their growth and development, such as growth regulators

and cell wall components. The second category is the secondary metabolites required for the

plant to enhance its ability to survive in its environment and overcome threats in its niche. In

other words, secondary metabolites are compounds produced in a plant that enables a plant to

adapt to its local environment. It is worth mentioning that secondary metabolites have various

functions in plant physiology and biochemistry. Any plant species propagating in an

unfavourable niche, such as tepid and aquatic tropical forests, will strive to preserve itself by

producing biomolecules that may have insecticide, fungicide, antibacterial, or antiviral

properties (Taha et al., 2019). However, assuming the leaves of a plant display no signal of

invasion, they may possess defensive bioactive molecules in opposition to insects and

microorganisms. The roots may often produce antifungal phytochemicals due to the rich

nature of pathogenic fungi in the soil. These secondary metabolites may also exhibit

antifungal reactions in opposition to human pathogenic fungi (Awuchi, 2019). Therefore, due

to the diverse functions that plant phytochemicals possess in plant cells, these molecules are

of unique interest to pharmacologists and biochemists. Suffice to say that specific secondary

metabolites are bioactive compounds evoking pharmacological or toxicological effects on

animals and humans. Bioactive phytochemicals can be broadly classified as terpenes and

terpenoids, alkaloids, and phenolic compounds (Rehab et al., 2018; Awuchi, 2019). Their

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specific structural characteristics hinge on the reaction pathway in which they were bio-

synthesized.

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1.3 Alstonia boonei Description

Alstonia boonei is a very large, deciduous, tropical-forest tree belonging to the Dogbane

Family (Apocynaceae). It is native to tropical West Africa, with a range extending into

Ethiopia and Tanzania. Its common name in the English timber trade is cheese wood, pattern

wood or stool wood while its common name in the French timber trade is emien (derived

from the vernacular of the Ivory Coast). The wood is fine-grained, lending itself to detailed

carving. Like many other members of the Apocynaceae (a family rich in toxic and medicinal

species), A. boonei contains alkaloids and yields latex.

Alstonia boonei is a tall forest tree, which can reach 45 metres (148 ft) in height and 3 m (9.8

ft) in girth, the bole being cylindrical and up to 27 m (89 ft) in height with high, narrow,

deep-fluted buttresses. On the Plateaux Batekes in Congo (Kinshasa) these trees have greatly

swollen bases like those of the Bald Cypress and Water Tupelo. The leaves are borne in

whorls at the nodes, the leaf shape is oblanceolate, with the apex rounded to acuminate and

the lateral veins prominent and almost at right angles to the midrib. The flowers are

yellowish-white and borne in lax terminal cymes. The fruits are pendulous, paired, slender

follicles up to 16 centimetres (6.3 in) long, containing seeds bearing a tuft of silky, brown

floss at either end to allow dispersal by the wind. The latex is white and abundant.

1.3.1: Medicinal Uses of Alstonia boonei (Stem Bark)

An infusion in cold water of the stem barks of Alstonia boonei is drunk as a cure for venereal

diseases, worms, snakebite and rheumatic pains and to relax muscles. An infusion of root and

stem bark is drunk as a remedy for asthma, a liquid made from the stem bark and fruit is

drunk once daily to treat impotence.

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Plate 1: Alstonia boonei

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1.4 Holarrhena floribunda Description

Holarrhena floribunda, commonly known as the false rubber tree, conessi bark or kurchi

bark, is a plant in the family Apocynaceae. Holarrhena floribunda grows as a shrub or tree up

to 25 metres (82ft), with a stem diameter of up to 30 centimetres (12 in). Its fragrant flowers

feature a white corolla. The fruit is pale grey to dark brown with paired follicles, each up to

60 centimetres (24 in) long.

1.4.1: Medicinal Uses:

Holarrhena floribunda is locally used in traditional medicine as a treatment for dysentery,

diarrhoea, fever, snakebite, infertility, venereal disease, diabetes and malaria. The stem bark

of Holarrhena floribunda is used traditionally as a febrifuge, tonic, as remedy for snakebite

and for the treatment of venereal diseases and dysentery. In some parts of west Africa, the

bark is used to treat abdominal pains, nausea, indigestion and diarrhea.

Plate 2: Holarrhena floribunda

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1.5 Aim of Study

This study was aimed at determining the effect of different extraction methods on the

phytochemical constituents of Alstonia boonei and Holarrhena floribunda (apocynaceae)

stem barks.

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 CHAPTER TWO

LITERATURE REVIEW

There are different Extraction methods on the phytochemical constituents of Alstonia boonei

and Holarrhena floribunda stem barks.

2.1 Maceration
Maceration is a simple extraction method that involves soaking the plant prepared raw

material in a coarse or powder form in a solvent of interest at room conditions for at least

three days with intermittent agitation (Azwanida, 2015). After the extraction is completed,

the mixture is strained either through sieves or a net with tiny holes. Subsequently, the marc

is pressed, and the liquid extract is cleaned using either filtration or decantation after

standing. Maceration is preferably carried out in a stoppered container to minimize solvent

loss through evaporation. It is undesirable to obtain an already concentrated extract through

evaporation of the solvent during the extraction process. The product is concentrated

frequently by the use of vacuum evaporation. It is crucial to select an appropriate solvent in

the maceration as the solvent will delineate the phytochemicals classes salvaged from the

samples. The solvent could also enable the extraction of thermolabile phytochemicals.

The procedure has the underlying disadvantage of low efficiency and long duration of

extraction (Azwanida, 2015). However, optimized conditions may attribute significant

efficiency to this technique as high phenolic compounds, and anthocyanins yield were

obtained from chokeberry (Ćujić et al., 2016). On the other hand, a study on C. cajan leaves

to extract flavonoids using microwave-assisted, reflux, ultrasound-assisted, and maceration

extractive methods observed that the maceration techniques afforded the lowest yield (Jin et

al, 2011). Flavonoids have also been extracted using maceration from rhizomes of turmeric

using nonionic surfactant Triton X-100 at a temperature of 35°Cat neutral pH, from fruits of

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Arbutus unedo L. at a raised temperature of 79.6°C in 3.7% diluted ethanol, and from leaves

of Ficus carica and Euphorbia neriifolia using 75% concentrated ethanol at room temperature

(Albuquerque, 2016). In these studies, the type of solvent used was based on the polarity of

the phytochemicals extracted. For instance, Triton X-100, which is nonionic but has a

hydrophilic side chain, was used to extract less polar flavonoids, whereas the more polar

ethanol, or a mixture of ethanol and water was used to extract more polar flavonoids Further,

Polyphenols, such as anthocyanins have been extracted from dried fruits of chokeberry using

50% ethanol as solvent (Ćujić et al., 2016).

2.2 Digestion
Digestion is an extractive method similar to maceration and uses slight warming in the

extraction process (Azwanida, 2015). Care is, however, exercised to avoid the temperature

altering the bioactive phytochemicals of given plant material. Therefore, there is increased

efficiency in using the extraction solvent due to warming. Mostly temperatures are kept in the

range of 35 to 40 °C but may be increased to a maximum of 50 ° C for tougher plant

materials such as barks and materials containing dismally soluble phytochemicals. During

extraction, desired plant parts are introduced in a container with the appropriate solvent pre-

heated to the indicated temperatures. The optimum temperature is maintained for a period

that may range from half an hour to 24 hours with shaking the container at regular intervals

(Azwanida, 2015).

2.3 Infusion and Percolation

Infusion is described as a dilute solution of easily soluble constituents of the plant material. It

is an extraction technique in which the plant material is immersed in boiling solvent,

particularly water, and left to stand in a stoppered container for about 15 minutes, after which

time the extract (tea) is poured off and separated from the marc using a filter (Azwanida,

2015). Tea may be considered as the best example of an infusion. For example, Caffeine has

15
been extracted from dried crushed leaves of tea brands alokozay, lipton, tapal, and tetley at

brewing times ranging from 2 to 30 minutes within the temperature range of 30 to 90 °C

(Sharif et al, 2014). Further, phenolic compounds were extracted from fruits of Tilia cordata

at an optimal temperature of 95 °C (Cittan et al, 2018). It has to be noted that some infusions

are prescribed to treat health issues such as diarrhea, bronchitis, and asthma. For instance,

antioxidants; phenols, and flavonoids have been extracted from rhizomes of various gingers

in boiling water for 10 minutes (Cittan et al, 2018). Another method of interest similar to

infusion but more efficient than maceration is percolation.

Percolation is the most popular procedure for preparing fluid extracts such as tinctures.

Percolation means "to pass a liquid through a solid material drop by drop." During

percolation, the solvent, commonly ethyl alcohol, is slowly passed through the plant material,

gradually packing itself with phytochemicals, and is gradually propelled down by another

fresh solvent added from the top (Azwanida, 2015). Before introducing plant material into the

percolator, it must be carefully shredded, not making the particles too small. If particles are

too fine, it will complicate separating the fine particles from the extraction solvent.

Consequently, the extract would be cloudy with residue settling at the bottom of the

percolator. Nonetheless, it is appropriate to moisten the plant matrix with the extraction

solvent, enabling the plant cells to elongate for smooth diffusion of phytochemicals into the

extraction solvent (Azwanida, 2015; Cittan et al., 2018].

After the plant material is inserted into the percolator, the extraction solvent is poured in from

the top and percolates through the plant material at a speed determined by the nature of the

plant material subjected to extraction. The solvent flow rate should not be excessive to allow

time for solvent penetration into plant cells and extract the constituent phytochemicals.

Nevertheless, the solvent percolation rate should not be too slow, leading to more solvent

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consumption for complete extraction. In general, for 1kg of plant material, the solvent flow

rate should be about 5mL per minute.

The choice of extraction solvent depends on the chemical attributes of the secondary

metabolites being extracted. A water-alcohol solvent mixture is commonly utilized, resulting

in extra efficient extraction because water hydrates plant walls as the alcohol is chemically

similar to most active components extracts from the plant material. For example, Phenolics,

particularly epicatechin, were extracted using 70% ethanol, and petroleum ether has been

used to extract the antioxidants such as phenols and flavonoids (Paz et al, 2017; Chanda et al,

2012). Interestingly, apart from alcohol, inorganic aqueous solution of hydrochloric acid was

used to extract alkaloids from wild fruits by percolation (Fei Zhang et al, 2005). However, the

alcohol has the added advantage of preserving the extract as it is a preservative. The extract is

termed leachate. After the process has ended, the plant material is pressed to recover the

solvent absorbed residually, and the residual solution is added to the leachate (extract).

Extraction ends upon elution of a colourless liquid, devoid of phytochemicals, from the

percolator (Azwanida, 2015).

2.4 Decoction
This extraction technique is useful for phytochemicals that do not decompose or modify with

increasing temperature. During decoction, plant material is boiled in water for 15 to 60

minutes (Azwanida, 2015). The duration of boiling will depend on the nature of plant tissues

and the phytochemicals being extracted. Ordinarily, delicate plant parts such as leaves, roots,

flowers, and tender stems are boiled for 15 minutes. For instance, phenols and flavonoids

have been extracted using decoction and infusion from fruits, rhizomes and leaves at 100 °C

(Chanda et al., 2012; Mahmudati et al., 2020). Instead, hard plant parts such as branches and

tree barks can be subjected to boiling for an hour. After boiling, the mixture is cooled and

17
then strained, adding cold water to obtain the required amount of solution. After the

decoction process is completed, the mixture is filtered to obtain the liquid extract.

The extract produced using the decoction technique is likely to have many undesirable

products. It may also be noted that it is not the ideal method for thermolabile compounds. It

has been reported that the bark extract of S. Cumini using decoction as an extractive

technique demonstrated significant antiglycation and antioxidant potential (Perera et al,

2017).

2.5 Soxhlet extraction

Soxhlet extraction is a continuous extraction of phytochemical constituents using a hot

solvent. The ground plant material is placed in a thimble (porous bag) made from either a

firm filter paper or cellulose (Azwanida, 2015). The thimble packed with ground plant

material is placed in the compartment of the Soxhlet paraphernalia. Extraction solvent such as

ethanol or methanol is placed in the bottom flask. The solvent is then heated and vaporized in

the sample thimble, is next condensed in the condenser on top of the apparatus, and then drips

back, resulting in the extraction of phytochemical constituents. The enhanced yield is

obtained compared to maceration-based extraction techniques. Fatty acids have been

extracted using this technique from hemp seeds at 70 °C for eight hours, and phenolic

compounds from leaves using 60 % ethanol for two hours extraction time (Rezvankhah et al.,

2019); Alara et al., 2018).

This extraction technique is quite efficient. Soxhlet extraction method afforded 38.21 mg g-1

of ursolic acid from traditional Chinese medicine cynomorium. However, because of the

higher temperature, this method risks the degradation of thermolabile compounds.

Interestingly, a study comparing Soxhlet and maceration extraction methods discovered that

the amounts of both polyphenols and alkaloid extracts decreased for the Soxhlet method

(Chin et al., 2013).

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2.6 Accelerated solvent extraction (ASE)

This technique has gained considerable importance because of its benefits like low solvent

demand, high output, and relatively less time consumed. The higher solvent temperature and

pressure mark the ASE operations' favourable condition. This technique is a more robust

solvent extraction technique than either maceration or Soxhlet extraction. There are examples

supporting significant ASE performance. For instance, it was found that ASE performed

better at recovering lipophilic and hydrophilic phytochemicals from raspberry pomace

compared with Supercritical Fluid Extraction (SFE). In ASE, the effects of temperature and

extraction time on extraction yield were significantly less but recovered 25% lipophilic and

hydrophilic compounds compared to the 15 % yield in SFE (Kryţ et al., 2016). In this

technique, the extraction cell made of stainless steel is packed with the sample, then filled

with solvent, and placed between inert silica layers separated with cellulosic filter papers.

The system is heated at an increased temperature and pressure for a pre-set time, the

conditions favour extraction due to increased diffusion coefficient and lowered viscosity. The

extract is collected in vials and cell cleaned by pumping fresh solvent and nitrogen. The inert

packing material prevents the plant matrix from forming aggregates that might block the

system (Rahmalia et al., 2015).

To attain enhanced phytochemical recovery yields, critical ASE extraction conditions include

but not limited to temperature, pressure, and extraction time. For example, the robustness of

ASE was assessed during the extraction of cocaine and benzoylecgonine from coca leaves.

The extraction technique was verified to be uniquely robust around an optimum temperature

of 80°C, the pressure of 20 MPa, and extraction time of 10 minutes (Brachet et al., 2001).

Interestingly, temperature variation significantly impacts the extraction efficiency of different

bioactive compound classes differently. For example, it was discovered that the highest

phenolic acids content yield was achieved with high temperature, whereas lower temperatures

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afforded more efficiency in extracting high yields of flavonoids (Gonçalves et al., 2018;

Figueroa et al., 2018).

It appears that the critical extraction conditions may influence extraction efficiency

individually or in combination. In some instances, a single parameter may affect extraction.

As was the case during the recovery of carbohydrates and phenolic compounds from barley

hull, pressure and extraction time were insignificant; the only temperature was found to

influence the extraction yield of phytochemicals (Suparna et al, 2014). In other

circumstances, some conditions may exhibit the combined influence of the extraction of

phytochemicals. For example, during the ASE of steviol glycosides from stevia leaves, it was

found that temperature, extraction time, and the number of cycles substantially affected

phytochemicals yield in a composite manner. At the optimum temperature of 100 °C, 4

minutes of extraction duration, and 1 cycle, an average of 91.8 ± 3.4% glycosides were

recovered. However, upon additional optimization of reducing the particle size of plant

samples to less than 0.5 mm the experimental extraction yield surged to 100.8 ± 3.3%

(Jentzer et al., 2015). In addition, during the extraction of carotenoids from food matrices,

time and temperature were found to have a significant effect on carotenoids' recovery yield.

The optimum conditions were temperature of 60 °C, extraction duration of 15 minutes in

three cycles. However, it was demonstrated that increasing the amount of solvent through a

number of cycles above three does not enhance further recovery of more carotenoids (Saha et

al., 2015). It may suffice to add that in comparison to some other conventional and modern

extraction techniques, ASE is very fast, very efficient, replicable, green, more convenient,

and does not require a lot of energy (Jentzer et al., 2015).

The phytochemical extraction yield is also specific to the nature of phytochemicals. Some

studies suggest that the extraction conditions can have opposite effects on the extraction yield

of two different types of phytochemicals. For instance, during the extraction of Beta-glucans

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and phenolic compounds from waxy barley using ASE, it was discovered that higher

temperature favoured lower extraction yield of the β-glucan, but higher recovery yield of

phenolics. The lower extraction yield of β-glucan could be attributed to the fragmentation of

the molecules at high temperatures (Óscar et al., 2015).

2.7 Microwave-assisted extraction

Microwave-assisted extraction (MAE), also known as microwave extraction, is a recent way

of extracting natural products that incorporate microwaves and solvents during the extraction

process. The Microwave frequency ranges from 300 MHz to 300 GHz. During the extraction

process, microwaves heat the solvent and plant tissue, enhancing the kinetics of extraction.

The microwaves heat the sample by directly impacting the polar molecules. The energy

conversion from microwave to heat involves dipolar rotations. Heating is directly

proportional to the dielectric constant of the solvents (Shashikant et al, 2002). The viscosity

of the solvent affects the extraction process significantly as lower viscosity facilitates the

dispersion of ions and hence solvation [47]. The extraction process involves the diffusion of

solvents into the sample, subsequent separation of solute from the functional site, and finally

releasing solutes to solvents. The technique is good at preserving the biological activities of

the extracts. For example, the optimization of MAE in green tea extraction confirmed the

improvement of the antioxidant activity of the phytocompounds and improved the total

phenolic content and the targeted colour quality of the extracts (Taşkın and Aksoylu, 2020).

Using the MAE technique, a variety of phytochemicals such as saponins have been obtained

from seeds, Polyphenolic antioxidants from leaves, sterols from dried mushrooms, and

flavonoids from leaves (Hu et al., 2021; Belwal et al., 2016; Heleno et al., 2016). Notably,

the phytochemicals such as flavonoids, polyphenols, and saponins extracted by MAE are

polar compounds, and microwaves directly impact these compounds, rendering the extraction

21
quite efficient. Several progressive and robust MAE instruments and methods are available,

such as solvent-free microwave-assisted extraction (SFMAE) and pressurized microwave-

assisted extraction (PMAE) (Ismail et al., 2021).

The heating of dried plant material targets the minute infinitesimally small traces of moisture

in plant cells. When microwaves heat the moisture inside the plant cell, evaporation ensues.

When evaporation occurs in dried plant cells, considerable pressure is developed on the cell

walls, thereby pushing the cell walls from inside. The cell wall thus elongates and eventually

raptures, thereby exuding the bioactive components. This process increases the yield of

phytochemicals (Ismail et al., 2021). Soaking the plant material in a suitable solvent before

MAE further yields. The solvent further facilitates the hydrolysis of glycosidic (ether) bonds

of cellulose into soluble fractions. The increased temperature also increases the

phytochemical yield, enhancing solvent penetration into cellular walls.

There is complete cell wall rupture in MAE as opposed to the heat–reflux extraction (HRE),

as revealed by scanning electron micrographs (SEM) of plant samples subjected to these

extraction techniques. In HRE, a series of solvent cellular penetration and phytochemical

solubilisation bring out the cells' bioactive compounds. On the other hand, phytochemicals

are exposed to the solvent through cell rapture in MAE. There is desorption of active

components from plant material in MAE. The infinitesimally small traces of vapour

occurring in plant glands and vascular tissues are heated, causing cell wall expansion leading

to phytochemicals flowing out of the cells. Therefore, the solvent and plant material dielectric

susceptibility affect the microwave energy used in MAE.

2.8: Ultrasound-assisted extraction, UAE (sonication extraction)

Ultrasounds are electromagnetic waves with higher frequencies than sound waves audible to

the human ear. The range of ultrasound utilized is from 20 kHz to 2000 kHz. It travels

22
through a medium involving expansions and contractions following the wave nature. The

mechanical effect of acoustic cavitation from the ultrasound increases the surface area of

contact between solvents and plant samples and the permeability of cell walls. The bubble

formation, its growth and collapse are termed as cavitation. Some studies observed that

frequency used can modify and favourably influence the extraction of compounds from the

sample (Machado et al., 2019).

Interestingly, a study observed the higher yields of phenolics at a lower frequency of 40 kHz

than at 120 kHz (González-Centeno et al., 2014). Such an observation prompts studies to

evaluate ultrasonic parameters simultaneously to enhance and optimize extraction efficiency.

A study using UAE reported phenols extraction from rhizomes with 75.3% ethyl alcohol over

the extraction time of 40 minutes higher yields compared to one with the solvent in context

(Wang et al., 2013). Ultrasonic-assisted extraction technique extracts bioactive compounds

by relying on ultrasound’s mechanical action on plant cell walls (Bimakr et al., 2017). The

mechanical action of ultrasound enhances the surface contact between solvent molecules and

the plant sample matrix. Thus, ultrasound modifies and disrupts plant materials’ physical and

chemical characteristics, expedites the release of phytochemicals, and further reinforces the

solvent system’s mass movement into plant cells (Julian et al, 2019; Altemimi et al., 2016).

The UAE technique was found to quicken the extraction process, lowered the energy

consumption, and increased the recovery of phytochemicals from annatto seeds. Most

importantly, UAE, in this study of annato, efficiently preserved the extracted phytochemicals

and thereby favouring the phytochemicals’ functional activities (Herrera et al., 2004)

The UAE is a crucial extractive technique for extracting bioactive compounds, evident from

its wide applications. Some phenolic compounds were reportedly extracted from strawberries

(Nishad et al., 2019) and oranges (Ghafoor et al., 2009), affording good results. Phenolic

derivative and anthocyanine extractions using grape-peel using this technique have also been

23
reported (Petigny et al., 2013). The efficiency of UAE is supported by a study reporting

considerably less time of 30 minutes and far better yields than the maceration method, which

took 120 minutes and afforded lower yields (Zhang et al., 2009). Furthermore, The UAE

method predominantly enhances the extraction speed and requires a relatively lower quantity

of the solvent (Oniszczuk and Podgórski, 2015; Navarro del Hierro et al., 2018). Thus, UAE

is an extractive method of marked preference. Using UAE, triterpenoid saponins were

obtained from edible seeds at optimal sonication output amplitude of 60% (Altemimi et al.,

2016), Phenolic compounds from slices of pumpkins at reduced ultrasonic power of 44.60%

(Ravanfar et al, 2018), and anthocyanins from leaves of red cabbage using an ultrasonic

output power of 100 W (Šic Ţlabur et al., 2021).

2.9 Supercritical fluid extraction (SFE)

The SFE technology has a broader application in extracting valuable compounds from

various sources at a commercial scale. This technique has great potential to extract valued

compounds from food products (Raja et al., 2021). The SFE may be defined by the changes

in the temperature and pressure required to convert gas into a liquid, where two phases are

not distinguishable. A Supercritical fluid substance shares both gas and liquid phases'

physical properties at its critical point (Raja et al., 2021). The critical region of a supercritical

fluid is determined by temperature and pressure. At the critical point, which is achieved

above critical temperature Tc and Critical pressure-Pc, gas and liquid phases become

indistinctive. The technique involves the solubilisation of extractable chemicals and their

separation. The solvent dissolves chemicals in the sample while flowing through the packed

bed. The solvent then leaves the extractor, and due to an increase in temperature and drop in

pressure, the extract becomes solvent free.

Though it leans more towards the gaseous character, a supercritical fluid has the solvating

properties of a liquid. Carbon dioxide, for instance, becomes supercritical at a temperature

24
above 31.1°C and 7380 kPa pressure. Several recent studies may be cited where supercritical

carbon dioxide has been used. For example, Oil lipids (consisting mainly of glycerol-lipids,

sterol esters, phospholipids) have been extracted from seeds at optimal Pressure of 80 MPa

and temperature of 57°C (Belo et al, 2019), alkaloids from seeds at a pressure of 27 MPa

(Rosas‐Quina et al, 2021), polyphenols, vitamins, anthocyanins, dietary phenolic compounds

and carotenoids from leaves of sweet cherry at a pressure of 30 MPa (Zhang et al, 2020),

essential oils from a herb at a Pressure of 30.4 MPa (Mostafa Khajeh et al., 2004), and

phenolics from potato peels at a pressure of 350 bar (de Andrade Lima et al., 2021). It is

pertinent to emphasize the low extraction temperature in SFE, which makes the technique

uniquely suitable for extracting thermolabile phytochemicals.

The use of Supercritical CO2 (scCO2) enhances extraction due to its strong solvation power

for nonpolar phytochemicals. However, polarized phytochemicals tend to have low solubility

in supercritical CO2. The solubility of polar phytochemicals in supercritical-CO2 can be

enhanced by adding a minute amount of ethyl alcohol or methanol, water, acetone, ethyl

acetate, acetonitrile, and the like. This adjustment increases the phytochemical yield of the

extract.

2.10: Enzyme-assisted extraction (EAE)

In certain plants, it is challenging to separate phytochemicals from the polysaccharide lignin

network stabilized by hydrogen bonding and hydrophobic interactions such as van der wall

forces. Phytochemicals in their matrices remain dispersed in the cell cytoplasm and are

inaccessible with the solvent extraction process. The bound phytochemicals in such samples

are effectively released at high yields by pre-treating the plant material with specific enzymes

(Calderón-Oliver and Ponce-Alquicira, 2021). These enzymes are added during extraction to

enhance phytochemical yield by breaking down cellular walls. Further, these enzymes

hydrolyze carbohydrates such as cellulose and lipid bodies. The specific enzymes used

25
include cellulase, pectinase, and amylase. Two main approaches that utilize enzymes during

extraction are enzyme-assisted aqueous extraction (EAAE) and enzyme-assisted cold

pressing (EACP). While the former technique has been used mainly to extract oils from

diverse seeds, the latter has been used to hydrolyze the plant's seed cellular wall (Calderón-

Oliver and Ponce-Alquicira, 2021). Using EAE, non-extractable polyphenols have been

obtained from fruits of sweet cherry at the optimal temperature of 55°C, carotenoids from

sunflower petals at 40°C, phenolics from citrus peels at temperatures varying from 20 to 60

◦C, lycopene from tomato peels at 30°C, flavonoids from grapefruit peels at 50 °C, and

anthocyanins from Cacahuacintle Maize (DomÃnguez-RodrÃguez et al., 2021; Li et al.,

2006; Antonio Zuorro et al., 2011; Zarate Vilet et al., 2020; Fernandez‐Aulis et al., 2019).

The EAE is utilized in conjunction with other extraction techniques as the enzymes make

non-extractable phytochemicals accessible to the solvent and hence vulnerable for extraction.

For example, the utilization of enzymes during microwave processing led to increased

extractability of phenolic compounds from olive pomace at higher extraction temperature and

faster heating strategy compared to low phytochemical recovery yield achieved by

conventional solvent extraction using water (Gabriela et al., 2021). Further, the sequential

treatment of sisal waste with enzymes followed by ultrasound led to higher yields of pectin

compared to low yields of pectin obtained by other extraction techniques without the

involvement of enzymes (Yang et al., 2017). The enzyme concentration and pH vary

depending on the enzymes’ nature and action. For instance, for carbohydrases, Viscozyme L,

a multi-enzyme complex containing arabinase, cellulase, β-glucanase, hemicellulase, and

xylanase, was used at optimum acidic pH of 4.5 and temperature of 50°C in a 0.1 M acetate

buffer to obtain the maximum extract yield (Habeebullah et al., 2019). It has also been

observed that enzyme concentration directly affects the yield of the extract. For example,

increased amounts of cellulase were found to enhance the licorice extraction yield (Giahi et

26
al., 2021). Pretreatment of plant material of interest is followed by an extraction method after

the enzymes detach the phytochemicals from plant material where they were extensively

bound. For example, flavonoids were obtained from Pomelo peels by first treating the peels

with 4.5% pectinase at various incubation times and afterward extracting the phytochemicals

using ultrasound-assisted extraction method at a reduced optimal temperature of 30 °C

(Nguyen et al., 2021). In the same vein, pretreating of the plant material such as rosemary

leaves with pectinolytic enzymes for 1 hour before a 24 hours solid-liquid conventional

extraction with 50% hydroethanolic solvent was found to be the optimum conditions for the

extraction of rosemary leaves, providing an extract with higher radical scavenging activity of

antioxidants than the corresponding extract without the enzyme pretreatment (Pontillo et al.,

2021).

27
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Plant Collection

Fresh stem barks of Alstonia boonei and Holarrhena floribunda were harvested from Student

Union Building (SUB), behind Mr Felix office, University of Nigeria, Nsukka. (UNN). They

were properly identified and authentication by Mr Felix Nwafor, a Plant taxonomist in the

Department Plant Science and Biotechnology, University of Nigeria, Nsukka.

3.2 Sample Preparation

They were washed in running tap water and were chopped into small pieces, and spread out

on a newspaper where they were allowed to dry in a well-ventilated room, at room

temperature (25 – 28 ⁰C). The dried samples were ground with a local milling machine and

stored separately in air-tight containers for further studies.

3.3 Extraction

3.3.1: Maceration

Twenty (20) grams each of dried and ground samples were macerated with 70% ethanol for

48 hours and filtered. Serial dilution was carried out where the extracts were measured up

to100ml with 70% ethanol and covered in air-tight containers.

3.3.2: Decoction

One (1) gram each of dried and ground samples were mixed with 70% ethanol and boiled for

nine minutes in a heating mantle and then filtered. Serial dilution was carried out where the

extracts were measured up to100ml with 70% ethanol and covered in air-tight containers.

28
3.3.3: Microwave Assisted Extraction (Mae)

One (1) gram each of dried and ground samples were mixed with 70% ethanol and heated

with microwave for three minutes and then filtered. Serial dilution was carried out where the

extracts were measured up to100ml with 70% ethanol and covered in air-tight containers.

3.4 Qualitative Phytochemical Screening

Qualitative phytochemical and quantitative phytochemical analysis of samples of the

powdered plant materials were performed according to the methods of Harborne (1973).

3.4.1: Detection of Alkaloids

The methanolic plant extract was warmed with 2% H 2SO4 for two minutes. It was filtered and

few drops of Dragendorff reagent was added. Formation of a reddish-orange precipitate

showed presence of alkaloids.

3.4.2: Detection of Terpenoids

Five milligrams of the extract were mixed with two millilitres of chloroform and

concentrated H2SO4 (3 ml) was carefully added to form a layer. An appearance of reddish-

brown colour in the inner face indicated the presence of terpenoids.

3.4.3: Detection of Flavonoids

Extracts were treated with few drops of sodium hydroxide solution. Formation of intense

yellow colour, which became colourless on addition of dilute acid indicated the presence of

flavonoids.

3.4.4: Detection of Phenolics

Ten (10) millilitres of the extracts were treated with few drops of ferric chloride solution.

Formation of bluish black colour indicated the presence of phenol.

29
3.4.5: Detection of Saponins

About 0.5 mg of the extract was shaken with five ml of distilled water. Formation of frothing

(appearance of creamy miss of small bubbles) which persisted after heating for 5 minutes

showed the presence of saponins.

3.4.6: Detection of Tannins

A small quantity of extract was mixed with water and heated on a water bath. The mixture

was filtered and ferric chloride was added to the filtrate. A dark green colour was formed. It

indicated the presence of tannins.

3.4.7: Detection of Carbohydrates

To 2 ml of the extract, 2 ml of Fehling’s solutions A and B were added and the test tube

containing the mixture was kept in a boiling water bath for 10 mins. Formation of red

precipitate confirmed the presence of carbohydrates.

3.5 Quantitative Phytochemical Analysis

Stock solutions of the samples were prepared by dissolving 100 mg of the extracts in 100 ml

of distilled water (1 mg/ml). These were used in quantitative determination of the present

phytochemical groups following the procedures of Madhu et al. (2016).

3.5.1: Estimation of Total Alkaloids

To one millilitre of methanolic extract 5 ml pH 4.7 phosphate buffer was added, this was

followed by addition of 5 ml BCG solution. The mixture was shaken and 4 ml of chloroform

was added. The lower chloroform layer was collected and place in a 10-ml volumetric flask

and then diluted to adjust volume with chloroform. The absorbance of the complex in

chloroform was measured at 470 nm against blank prepared as above but without extract.

30
Atropine is used as a standard material. The total alkaloid concentration was read from an

atropine standard curve. Results were expressed as µg atropine equivalents.

3.5.2: Estimation of Total Terpenoids

To one millilitre of the plant extract, 3 ml of chloroform was added. The sample mixture was

thoroughly vortexed and left for 3 minutes and then 200 μl of concentrated sulfuric acid

(H2SO4) was added. Then it was incubated at room temperature for 2hours in dark condition

and during incubation a reddish-brown precipitate was formed. Then carefully and gently, all

supernatant of reaction mixture was decanted without disturbing the precipitation. 3 ml of

95% (v/v) methanol was added and vortexed thoroughly until all the precipitation dissolve in

methanol completely. The absorbance was read at 538 nm using UV/visible

spectrophotometer. The total terpenoid content was calculated by calibration curve of

Linalool and the results were expressed as Linalool equivalent (mg/g).

3.5.3: Estimation of Total Saponins

Vanillin–Sulfuric acid assay was used to determine saponin content of the extract. 0.5 ml of

aqueous sample solution, 0.5 ml vanillin solution of 8% (w/v) and lastly 5.0 ml of sulfuric

acid of 72% (w/v) were added and mixed in an ice water bath. After the mixture was then

warmed in a bath at 60˚C for 10 minutes then cooled in ice–cold water, the absorbance was

measured at 527 nm. Saponin concentration was calculated from a calibration curve. As a

saponin standard, oleanolic acid was used and results were expressed as µg oleanolic acid

equivalents

3.5.4: Estimation of Total Phenolic Content (TPC)

To 1 ml of the extract and 9 ml of distilled water, 1 ml of Folin-Ciocalteu reagent (1N) was

added shaken. The tube was incubated at room temperature for 5 minutes. To this was added

10 ml of 7% NA2CO3 solution and the volume was made 25 ml with distilled water. Gallic

31
acid standard solution was prepared in different concentrations (20, 40, 60, 80, 100 and 200

ug/ml) and treated as the samples. These were incubated for 90 min at 30 ºC. The absorbance

was read at 550 nm with UV-V is spectrophotometer. The total phenolic concentration was

read from the gallic acid standard curve and expressed as mg gallic acid equivalents (GAE)/g

of extract.

3.5.6: Estimation of Total Flavonoids Content (TFC)

The total flavonoids content was estimated using the aluminium chloride method. 1.0 ml of

plant extract was diluted with 200 µl of distilled water followed by the addition of 150 µl of

sodium nitrite (5%) solution. This mixture was incubated for 5 min and then 150 µl of

aluminium chloride (10%) solution was added and allowed to stand for 6 min. Then 2 ml of

sodium hydroxide (1 M) solution was added and made up to 5 ml with distilled water. The

mixture was shaken well and left it for 15 minutes at room temperature. The absorbance was

measured at 510 nm. Appearance of pink colour showed the presence of flavonoids content.

The total flavonoids content was expressed as quercetin equivalent (mg RE/g) of the extract

from the standard curve.

3.5.7: Estimation of Total Tannins Content (TTC)

Exactly 0.1 ml of the plant extract or standard was mixed with 0.5 ml Folin’s phenol reagent

and 7.5 ml of distilled water. Then 1 ml of 35% sodium carbonate was added the mixture and

made up to 10 ml with distilled water. The mixture was shaken and allowed to stand for 30

minutes at 30 ºC. The blue colour produced was read at 640 nm using UV/visible

spectrophotometer. The tannin content was calculated by calibration curve of tannic acid and

the results were expressed as tannic acid equivalent (mg TA/g) of the extract.

3.5.8: Estimation of Total Carbohydrates

32
For estimating the polysaccharide content, take 1ml of sample solution and add 1 ml of 5%

phenol and then add 5 ml of concentrated sulphuric acid mix well and leave for 10 minutes.

Measure the absorbance at 488 nm against blank. Then compare it with standard solution of

glucose. To prepare blank, 1 ml of distilled water added to1 ml of 5% phenol followed by 5

ml of concentrated sulphuric acid.

3.6: Statistical Analysis

The data obtained from the analyses were processed. Statistical package for social
science (SPSS version 22.0) was used to analyse the results obtained from the
phytochemical tests. Duncan Multiple Range Test (DMRT) and Analysis of variance
(ANOVA) were used for mean separation.

33
 CHAPTER FOUR

RESULTS

Three different extraction methods such as maceration, Decoction and microwave assisted
extraction were performed to determine the effect of different extraction methods on the
phytochemical constituents of Alstonia boonei and Holarrrhena floribunda stem barks. The
extracts were subjected to a phytochemical screening to determine the presence of
phytochemicals and its amounts.

The results are shown below.

4.1 Qualitative Analysis

Three methods of extraction such as maceration,Decoction and microwave assisted extraction

methods were performed and a qualitative test was carried out on the samples and the results
were obtained.

The table below shows the qualitative test carried out on the stem bark of Alstonia boonei.It
indicates the presence of alkaloids,flavonoids,phenolics,tannins,saponins,and soluble
carbohydrates.

Table 1: Qualitative analysis on Alstonia boonei stem bark.

Phytochemicals Maceration Decoction MAE

Alkaloidsmg/g Present Present Present

Flavonoidsmg/g Present Present Present

Phenolicsmg/g Present Present Present

Tanninsmg/g Present Present Present

Saponinsmg/g Present Present Present

Soluble Present Present Present

Carbohydratemg/g

34
Three different extraction methods were also performed and a qualitative test was carried out

on Holarrhena floribunda stem bark. In the table below, it indicates the absence of alkaloids

but flavonoids, phenolics, tannins, saponins, and soluble carbohydrates were present after the

qualitative test.

Table 2: Qualitative analysis on Holarrhena floribunda Stem bark

Phytochemicals Maceration Decoction MAE

Alkaloids Absent Absent Absent

Flavonoids Present Present Present

Phenolics Present Present Present

Tannins Present Present Present

Saponins Present Present Present

Soluble Carbohydrate Present Present Present

35
4.2 Quantitative Analysis

Quantitative analysis was carried out on the samples and a visible spectrophotometer was
used to determine the amount of these phytochemicals: alkaloids, flavonoids, Phenolics,
tannins, saponins and soluble carbohydrates.

The table 3 below shows the quantitative phytochemical analysis carried out on Alstonia
boonei stem bark.From the results below, alkaloid test gave 5.00±0.33 for maceration,
alkaloid test gave 5.33±1.00 for decoction, alkaloid test gave 4.33±0.33 for microwave
assisted extraction.From this,decoction has more of alkaloids than maceration and Mae.
Flavonoid gave o.300±0.33 for maceration,flavonoid gave 0.57±0.67 for decoction,flavonoid
gave 2.75±0.15 for Mae.from the results,microwave assisted extraction has more of
flavonoid. For phenolics,decoction has more of phenolics than maceration and Mae. For
tannins, decoction extracted more of tannins than the other two. For saponins,Mae extracted
more of saponins than the other two. For soluble carbohydrates,Mae has more of soluble
carbohydrates than others.

Table 3: Quantitative Phytochemical analysis on Alstonia boonei stem bark

Phytochemicals Maceration (mg/g) Decoction MAE (mg/g)

(mg/g)

Alkaloids 5.00 ± 0.33a 5.33 ± 1.00a 4. 33 ± 0.33a

Flavonoids 0.300 ± 0.33b 0.57 ± 0.67b 2.75 ± 0.15a

Phenolics 0.74 ± 0.00a 0.89 ± 0.18b 0.07 ±0.03a

Tannins 0.14 ± 0.14a 1.23 ± 0.14b 0.54 ± 0.39ab

Saponins 0.43 ± 0.56c 6.38 ± 0.00b 8.99 ± 0.39a

Soluble Carbohydrate 1.95 ± 0.45b 4.06 ± 1.02b 9.90 ± 1.73a

36
From the quantitative test below, alkaloid gave 0.00±0.00 for maceration, decoction and Mae.
For flavonoid Mae has more of flavonoid. For phenolics, Mae extracted more of phenolics.
For tannins, maceration extracted more of tannins than decoction and Mae. For saponins,
Mae has more of saponins than others. For soluble carbohydrates, Mae extracted more of
soluble carbohydrates than maceration and decoction.

Table 4: Quantitative Phytochemical Analysis on Holarrhena floribunda Stem Bark

Phytochemicals Maceration (mg/g) Decoction MAE (mg/g)

(mg/g)

Alkaloids 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Flavonoids 2.58 ± 0.18b 1.67 ± 0.10c 4.78 ± 0.12a

Phenolics 3.25 ± 0.30a 3.21 ± 0.25a 3.75 ± 0.27a

Tannins 8.98 ± 1.69a 7.46 ± 1.15a 7.50 ± 0.24a

Saponins 0.43 ± 0.56b 0.54 ± 0.06ab 0.99 ± 0.17a

Soluble Carbohydrate 2.99 ± 0.18b 10.63 ± 0.18a 12.29 ± 0.70a

37
 CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.1: Discussion

Phytochemical constituents, which are bioactive compounds found in different parts of plant

have been observed on the phytochemical analysis on the stem barks of Alstonia boonei and

Holarrhena floribunda using different extraction methods such as maceration, decoction and

microwave assisted extraction methods. The phytochemical constituents of Alstonia boonei

and Holarrhena floribunda stem barks have proved that they are of great important and are

highly medicinal as they help in the treatment of venereal diseases, asthma, snakebite,

rheumatic pains, dysentery, impotence and malaria.

From the results obtained, it has shown that different extraction methods produce different

values of phytochemical constituents of the stem barks of Alstonia boonei and Holarrhena

floribunda which includes flavonoids, phenolics, alkaloids, tannins and saponins which have

different techniques and processes of extraction. The results obtained from the qualitative

phytochemical and quantitative phytochemical analysis have shown the presence of some

phytochemicals and their amounts. It was also observed that the phytochemical constituents

of the stem barks of the two plants may have similar or different roles in human health. It was

also observed that some factors are considered during phytochemical extraction.They include

the nature of the plant material,choice of solvent,amount of the material,heating

temperature,extraction methods,and duration of extraction.These factors may affect the

quality and quantity of the extract.The choice of solvent is crucial during phytochemical

extraction and some of the important factors to consider are its

volatility,toxicity,corrosivity,viscosity,and chemical stability.The solvent used for extraction

process,for instance,should have high volatility so that it can be easily removed from the

38
extract.The solvent should be non-toxic or have a very low toxicity.The solvent should not be

too expensive and have low corrosivity.The desired compound to be extracted should be

soluble in the selective solvent.It should have a less boiling point in comparing to solute,that

is,easy to separate out.It was also observed that some solvents are used for for the extraction

of different classes of compounds and the solvents include

water,ethanol,methanol,chloroform,ether and acetone.Water is used for the extraction of

anthocyanins,starches,tannins,saponins,terpenoids,polypeptides,and lectin.Ethanol is used for

the extraction of tannins,polyphenols, polyacetylenes, flavonols, terpenoids, sterols and

alkaloids. Methanol is used for the extraction of anthocyanins, terpenoids, saponins, tannins,

xanthoxylene, totarol, quassinoids, lactones, flavones, phenones and polyphenols.

Chloroform is used for the extraction of terpenoids and flavonoids. Ether is used for the

extraction of alkaloids, terpenoids, coumarins and fatty acids. Acetone is used for the

extraction of phenol and flavonols. It was observed that maceration method is simple and

suitable for extracting water-soluble or alcohol soluble phytochemicals.Maceration can be

time-consuming and may result in a low extraction yield.Decoction method is useful for

phytochemicals that do not decompose or modify with increasing temperature.Microwave -

assisted extraction is a modern technique used to extract phytochemicals from plants. It

harnesses the power of microwave energy to enhance the extraction process.The microwave

energy promotes the release of phytochemicals from the plant material into the

solvent.Microwave assisted extraction significantly reduces the extraction time compared to

traditional methods.The application of microwave energy improves the solubility and

diffusion of phytochemicals, leading to higher extraction yields.The selective heating of the

plant material enhances the extraction efficiency while preserving the integrity of heat-

sensitive compounds.

39
5.2: Conclusion

There is considerable attention toward natural product research globally. The extraction of

phytochemical constituents of Alstonia boonei and Holarrhena floribunda stem barks is a

critical step in natural product research. Currently, extraction involves separating medicinally

active molecular components of plant tissues from the inert components by using traditional

solvent extraction techniques or the standard modern and green extraction procedures. The

extraction technique’s choice is crucial as it determines the reliability and quality of

subsequent analytical activities. The main focus of extraction is to achieve economic

viability, environmentally friendliness, shorter extraction time, and better yields of

phytochemical constituents without compromising the biological activities. As the

phytochemical constituents of Alstonia boonei and Holarrhena floribunda stem barks are

highly medicinal, which help in curing of venereal diseases, snakebite, rheumatic pains,

asthma, dysentery, infertility, diabetes and malaria, their different extraction methods should

be carefully followed and performed.

40
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