CHAPTER ONE

1.0 INTRODUCTION

A medicinal plant is defined as any plant species that contain secondary metabolites that

can be used as treatment for diseases or can be used as precursors for the manufacture of novel

therapeutics. It is a plant that contains one or more of its organs, materials that can be used for

treatment purposes or primary materials to form useful drugs. Medicinal plants are commonly

used because it is easy to get at the tips of the fingers and it’s less expensive (Manap et al. 2019).

There are different phytochemicals present in medicinal plants the common ones are

glycosides, tannins, alkaloids, soap, flavonoids and on the other hand, the least prevalent

compounds were resin and they are distributed in plant in this proportion glycosides (82 %, 70

species), tannins (68 %, 58 species), alkaloids (56 %, 48 species), soap (52 %, 44 species) and

flavonoids (35 %, 30 species) and resin (31 %, 26 species) (Samiha et al. 2022).

Many plants species are abundantly used to treat various diseases associated with skin

and stomach diseases, respiratory infections, tuberculosis, infections, ansarca, cancer,

astringents, spasms, coughs, convulsions, diarrhea, dysentery, headaches, high blood pressure,

snakebites e.t.c. (Ullah et al., 2020).

1.1 STATEMENT OF THE PROBLEM

The number of individuals suffering from one or more metabolic disorders or infectious disease

worldwide has been documented from different studies to be high (Okarfo 2012). Many

allopathic drugs used in management of the ailment are expensive in many third world and

developing countries (Akintunde et al,. 2011) . On the same hand, many of these drugs elicit

effects that are undesired by the patients. Due to the aforementioned, herbs and natural products

with folkloric medicinal importance are employed in the management of these ailments.

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Unfortunately, many of these medicinal plants are consumed without dosage and caution and this

phenomenon has been highlighted to be a cause of public concern and damage to vital organs

like liver.

1.2 JUSTIFICATION OF THE STUDY

Allium sativum and [Link] are two plants which are locally prescribed and commonly

consumed in combination in the management of elevated blood pressure in South-western

Nigeria. Therefore, this study was designed at evaluating the effect of Allium sativum and

[Link] extract on liver function of male Wistar rats.

1.3 AIM

The aim of this study is to evaluate the effect of Allium sativum L and Bryophylum

pinnatum Lam. liver function test of male Wister rat.

1.3.1 OBJECTIVES

(i) Carry out methanol extraction of Alium sativum L

(ii) Carry out methanol extraction of Bryophylum pinnatum Lam

(iii) Determine the acute toxicity and lethal dosage (LD50) of Allium sativum and Bryophyllum

pinnatumm methanol extracts

(iv) To evaluate the effect of the extract on liver function in male Wistar rats

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 CHAPTER TWO

2.0 LITRATURE REVIEW

2.1 Allium sativum L. (GARLIC)

Allium sativum L. (Figure 1) belongs to the Amaryllidaceae (Table 1) family of plants

commonly known as garlic is an aromatic herbaceous annual spice and one of the oldest

authenticated and most important herbs that have been used from ancient times as traditional

medicine (Badal et al., 2019). It is considered the second broadly used Allium species with onion

(Allium cepa L.), which is used as a remedy against several common diseases such are cold,

influenza, snake bites, and hypertension (Mukthamba et al., 2017).

Figure 2.1; Allium sativum L. obtained from Jagun Market Ogbomoso Oyo State

Table 2.1 Taxonomical Classification of A. sativum L.

Kingdom Plantae

Class Tracheophytes

Subclass Angioasperms

Order Asparagales

Family Amarylidaceae

Subfamily Allioideae

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 Genus Allium

2.1.1 Botanical Description of Allium sativum L.

Allium sativum is a perennial flowering plant that grows from a bulb. It has a tall, erect flowering

stem that grows up to 1 m (3 ft) (Takashima et al., 2017). The leaf blade is flat, linear, solid, and

approximately 1.25–2.5 cm (0.5–1.0 in) wide, with an acute apex (Takashima et al., 2017). The

bulb has a strong odor and is typically made up of 10 to 20 cloves (Bopda et al., 2014). The

cloves close to the center are symmetrical, and those surrounding the center can be asymmetrical.

Each clove is enclosed in an inner sheathing leaf surrounded by layers of outer sheathing leaves

(Maria et al., 2020).

2.1.2 Geographical Distribution of Allium sativum L. (GARLIC)

Allium sativum L. has its primary center of origin in central Asia (Kazakhstan) and the

Mediterranean and Caucasian zones are considered as the secondary centers (Haiping et al.,

2013). China produces 76% of the world's supply of garlic (Haiping et al., 2013). In Nigeria

garlic is grown commercially in the Northern regions which include Sokoto, Borno, and Kano

under irrigation during the dry season between the months of November to March when the

temperature is low (Suleyman et al., 2017).

2.1.3 Phytochemical Constituents OF Allium sativum L. (GARLIC)

Bulbs of A. sativum L. are reported to contain hundreds of phytochemicals some of which

chemical structure is shown in (Figure 2). Which is including sulfur-containing compounds such

as ajoenes (E-ajoene, Z-ajoene), thiosulfinates (allicin), vinyldithiins (2-vinyl-(4H) -1,3-dithiin,

3-vinyl-(4H)-1,2-dithiin), sulfides (diallyl disulfide (DADS), diallyltrisulfide (DATS)) and

others that accounted 82% of the overall garlic sulfur content (Al-Snafi 2013). Alliin, the main

4
cysteine sulfoxide is transformed to allicin by allinase enzyme after cutting off the garlic and

breaking down the parenchyma . S-propyl-cysteine-sulfoxide (PCSO), allicin and S-methyl

cysteine-sulfoxide (MCSO) are the main odoriferous molecules of freshly milled garlic

homogenates (Zeng et al., 2017). PCSO can produce more than fifty metabolites depend on

water content and temperature as well as allinase enzyme that can act on the mixture of MCSO,

PCSO, and alliin to produce other molecules, such as allyl methane Nutrients 2020, 12, 872 3 of

21 thiosulfinates, methyl methanethiosulfonate, and further corresponding thiosulfinates (R-S-S-

R0 ), by which R and R0 are allyl, propyl, and methyls groups (Zeng et al., 2017).

S-alk(en)yl-l-cysteine sulfoxides are the secondary metabolites obtained from cysteine which

accumulate in the plants of Allium genus (Souza et al.,2011). Garlic formulations consist of

several organosulfur compounds, N-acetylcysteine (NAC), S-allyl-cysteine (SAC) (Asdaq et al.,

2011) and S-ally-mercapto cysteine (SAMC), which are derived from alliin (Tran et al., 2018).

Notably, SAC has antioxidant, anti-inflammation, regulated redox, pro energetic, antiapoptotic,

and signaling capacities (Souza et al.,2011), while SAMC shows an anticancer activity through

preventing the cancer cells multiplication (Cao et al.,2017). Allicin (allyl thiosulfinate), is a

sulfenic acid thioester and its pharmacological effect is attributed to its antioxidant activity as

well as its interaction with thiol-containing proteins (Borlinghaus et al.,2014). In the allicin

biosynthesis, cysteine is transformed to alliin that is hydrolyzed by the allinase enzyme. This

enzyme composed of pyridoxal phosphate (PLP) which splits alliin and produces ammonium,

pyruvate, and allyl sulfenic acid that are highly reactive and unstable at room temperature, where

two molecules were combined to form allicin (Borlinghaus et al.,2014).

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Figure 2.2: Structures of phytochemicals identified in A. sativum L.

2.1.4 Pharmacological and Biological Activity of Allium sativum L.

[Link] Antioxidant activity of Allium sativum L.

Asdaq et al., (2011), reported that the frequent garlic intake promotes internal antioxidant

activities and reduces oxidative adverse effects either by increasing the endogenous antioxidant

synthesis or reducing the production of oxidizers such as oxygen-free radical species (ORS).

Gentamycin is an antibiotic that has been used to treat several types of bacterial infections and

was reported to promote hepatic damage through raising aspartate transaminase and alanine

aminotransferase enzymes in addition to lowering the plasma albumin level. It is demonstrated

that garlic protects against gentamycin- as well as acetaminophen-induced hepatotoxicity by

improving antioxidant status, and regulating oxidative stress (Wallock et al., 2014). Alliin, the

major compound isolated from aqueous garlic extract, showing wide-spectrum antioxidant

activities by controlling ROS generation and preventing mitogen-activated protein kinase

6
(MAPK). Moreover, it was reported to prevent ROS production by inhibiting NADPH oxidase 1,

and thus, inhibiting the osteoclast fusion caused by receptor activator of nuclear factor-kappa B

ligand (RANKL) (Liu et al., 2018). Allicin, diallyldisulfide and diallyltrisulfide are the main

antioxidative compounds that showed an antioxidant effect in lower doses at the physiological

level (Wallock et al., 2014).

[Link] Anti-inflammatory activity of Allium sativum L.

Garlic extracts and its related phytochemicals have been reported to possess anti-inflammatory

activity (Jain et al., 2015) A study reported that the garlic extracts remarkably impaired the liver

inflammation and damage caused by Eimeria papillate infections (Ahmad et al.2016). Gu et al.,

(2013), observed that the anti-inflammatory activity of garlic is caused by inhibiting the

emigration of neutrophilic granulocytes into epithelia. Aged black garlic (ABG) exhibited potent

antioxidant activities and these activities may be responsible for its anti-inflammatory activity.

Another report indicated that thiacremonone (a sulfur compound isolated from garlic) prevents

neuroinflammation and amyloidogenesis by blocking the NF-κB activity, and therefore can be

used to treat neurodegenerative disorders (e.g., Alzheimer’s disease) related to inflammation (Jin

et al.,2013).

[Link] Antibacterial activity of Allium sativum L. fresh extract and powder

Several studies have evaluated the antibacterial activity of various garlic preparations such as

crude or fresh garlic extract (FGE), and garlic paste (Asdaq et al., 2011) . The antibacterial

activity of garlic paste and FGE against commensal and pathogen enteric bacteria such as

Escherichia coli, E. coli O157:H7, Salmonella species, Shigella species, Vibrio species,

Campylobacter species, Listeria monocytogenes, Enterobacter, and Enterococcus species,

Lactobacillus acidophilus, Staphylococcus aureus, Streptococcus species, and Clostridium

7
difficile has been reported by various laboratories (Lu et al., 2011b; Roshan et al., 2017). In

addition, various studies have evaluated the impact of garlic and its organosulfur compounds on

the gut microbiome. Garlic was found to positively influence the gut microbiome and protect the

gut microbiome damage from high-fat diet (Chen et al., 2019). The gut microbiome was altered

upon intragastric administration of DADS of rat, a low dose of DADS decreased Bacteroidetes

phyla but increased Firmicutes phyla bacteria (Yang et al., 2019).

There are several reports of antibacterial activity of aqueous garlic extract (AGE) against a

variety of bacteria. Different studies reported that AGE exhibited activity against a large variety

of Gram-positive and Gram-positive pathogenic bacteria including MDR strains and isolates

such as MDR M. tuberculosis showing not only the effectiveness of garlic against drug-resistant

bacteria but also its broad spectrum (Gull et al., 2012; Meriga et al., 2012). In an interesting

study, it was found that counts of S. aureus in hamburger upon addition of aqueous garlic extract

reduced in a dose-dependent manner during storage for different time points in the fridge and

freezer, supporting the idea of using garlic for meat preservation (Mozaffari Nejad et al., 2014).

The extracts exhibited some degree of antibacterial activity against test enteropathogenic

bacterial strains with Salmonella enteritidis being the most sensitive. The tested bacteria were

more sensitive to ethanolic extract compared to other extracts (Pavlovic et al., 2017). Allicin

along with other thoisulfinates present in EGE seems to be responsible for its antibacterial

activity. Other than ethanol and methanol extract, the chloroform extract of both aged and non-

aged garlic exhibited activity against B. cereus by disk diffusion assay (Jang et al., 2018). Allicin

along with other thoisulfinates present in EGE seems to be responsible for its antibacterial

activity. Other than ethanol and methanol extract, the chloroform extract of both aged and non-

aged garlic exhibited activity against B. cereus by disk diffusion assay (Jang et al., 2018).

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Garlic oil is obtained by steam distillation of macerated or mashed garlic. A recent study has

performed an exhaustive analysis of the content of galic oil and reported that the majority of

garlic oil is composed of diallyl and allyl methyl sulfides (Mnayer et al., 2014). The anti-

mycobacterium effect of garlic oil was demonstrated using in vitro and in vivo studies

(Viswanathan et al., 2014). However, disk diffusion assay found garlic oil to be effective against

S. aureus, E. coli, P. aeruginosa, B. subtilis, and MRSA (Casella et al., 2013; Viswanathan et al.,

2014). Other bacteria that were reported to be sensitive to garlic oil are Salmonella typhi, L.

monocytogenes, and Campylobacter jejuni (Robyn et al., 2013; Mnayer et al., 2014).

Allicin can react with itself to yield ajoene, which is found abundantly in oil-macerated garlic.

Besides, two ajoene-related compounds Z-10-devinylajoene and iso-E-10-devinylajoene were

also isolated from oil-macerated garlic extract. Mice challenged with P. aeruginosa cleared the

infection rapidly when treated with ajoene compared to the control group (Jakobsen et al., 2012).

Pure ajoene exhibited antibacterial activity against Cronobacter sakazakii in a concentration-,

time-, and temperature-dependent manner (Feng et al., 2014). More studies testing the activity of

ajoene against more bacteria, especially clinical isolates and MDR strains, along with stability

and pharmacokinetic studies are needed to better understand and utilize ajoene and its related

compounds as antibacterial agents.

The major constituents of garlic organosulfide are various aliphatic disulfides. DADS, which is

the most abundant allyl sulfides in garlic organosulfide. DAS exhibits poor anti-H. pylori effect,

but this activity increased as the number of sulfurs increased. A study using disk diffusion assay

reported that DADS was effective, whereas dipropyl disulfide was not effective against S.

aureus, E. coli, and P. aeruginosa (Casella et al., 2013). In vitro studies reported that DAS,

DADS, and DATS exhibit activity against C. jejuni (Lu et al., 2011a; Robyn et al., 2013).

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Mixtures of DASs with various amounts of mono- to hexasulfides were prepared, and their anti-

mycobacterial activity was evaluated. It was found that while all the combinations exhibited

some activity, the most potent combination was the one that had higher quantity of DATS

(Oosthuizen et al., 2017).

It is an established fact that allicin is an effective, broadspectrum, and principal antibacterial

component of garlic. A meta-analysis of clinical data indicated that adding allicin to

conventional therapy improves the eradication of H. pylori infections (Si et al., 2019). Allicin

along with related thoisulfinates, allyl methyl, and methyl allyl thiosulfinate were found and

purified from acetone garlic extract. In a recent report, allicin was found to be active against C.

difficile and other commensal gut bacteria, and no significant synergy was observed when allicin

was tested with standard antibiotics (Roshan et al., 2017). The same group reported that allicin

did not affect spore germination, but significantly inhibited spore outgrowth of C. difficile spores

(Roshan et al., 2017).

[Link] Anti-cancer activity of Allium sativum L.

A 2016 meta-analysis of case-control and cohort studies found a moderate inverse association

between garlic intake and some cancers of the upper digestive tract (Guercio et. al 2016).

Another meta-analysis found decreased rates of stomach cancer associated with garlic intake, but

cited confounding factors as limitations for interpreting these studies (Zhou et al., 2011). Further

meta-analyses found similar results on the incidence of stomach cancer by

consuming allium vegetables, including garlic (Kodali et al.,2015). A 2014 meta-analysis

of observational epidemiological studies found that garlic consumption was associated with a

lower risk of stomach cancer in Korean people (Woo et al., 2014).

[Link] Anti –hypertensive activity of Allium sativum L.

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As of 2016, clinical research found that consuming garlic produces only a small reduction

in blood pressure (4 mmHg), and there is no clear long-term effect on hypertension,

cardiovascular morbidity or mortality (Varshney et al., 2016).

Garlic can also reduce oxidative stress, increase the production of Nitric oxide and

hydrogen sulfide (H2S), and inhibit the angiotensin converting enzyme, thereby lowering

hypertension ( Fasolino et al., 2015). A study showed that AGE could stimulate the production

of Nitric oxide, leading to endothelial-dependent vasodilation in the isolated rat aortic rings.

Moreover, L-arginine in aqueous garlic extract was crucial in the NOS-mediated Nitric oxide

production (Takashima et al., 2017). In addition, S-1-propylenecysteine was shown to be the key

antihypertensive compound. S-1-propylenecysteine was shown to improve peripheral blood

circulation and reduce the systolic blood pressure in spontaneous hypertension rats, without

affecting the systolic blood pressure of control rats (Ushijima et al., 2018).

In another study, the nitrites in the fermented garlic extract (FGE) could be converted into Nitric

oxide in vivo by Bacillus subtilis. Further, Nitric oxide reduced the systolic blood pressure in

spontaneous hypertension rats through the soluble guanylyl cyclase (sGC)-cyclic guanosine

monophosphate (cGMP)-protein kinases G (PKG) pathway (Park et al., 2016). Also, fermented

garlic extract was shown to alleviate pulmonary hypertension by decreasing the expression of

vascular endothelial cell adhesion molecule-1 and matrix metalloproteinase-9 (MMP-9) and

increasing the expression of PKG and endothelial nitric oxide synthase (eNOS) in

monocrotaline-induced pulmonary hypertension rats (Park et al., 2017). The anti-hypertensive

mechanisms of garlic are shown in Figure 3

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Figure 2.3; The mechanisms of the antihypertensive properties of A. sativum L.(Park et al.,

2017).

Garlic increases the production of nitric oxide (NO) in vascular smooth muscle cells. The L-

arginine (L-Arg) in aqueous garlic extract (AGE) could be transformed into NO and L-citruline

(L-Cit) mediated by nitric oxide synthase (NOS) (Han et al., 2011). Moreover, the nitrite in the

fermented garlic extract (FGE) could be converted into NO in vivo by Bacillus subtilis. NO and

atrial natriuretic peptide (ANP) activated particulate guanylyl cyclase (pGC) and soluble

guanylyl cyclase (sGC), thus catalyzing the transform of guanosine triphosphate (GTP) to cyclic

guanosine monophosphate (cGMP) (Sohn et al., 2012) . The elevated cGMP activated PKG, and

PKG decreased intracellular Ca2+ concentration by increasing intracytoplasmic Ca 2+ transport

into the sarcoplasmic reticulum through the sarco/endoplasmic reticulum Ca 2+-ATPase (SERCA)

pathway, thereby preventing the release of Ca2+ from the sarcoplasmic reticulum to the

12
cytoplasm, and stimulating the Ca2+-activated K+ (BKCa) channel on the cell membrane, as well

as reducing the Ca2+ influx (Takashima et al., 2017). As a result, the vascular smooth muscle

relaxed, and the blood vessels dilated.

[Link] Anti-Inflammatory Activity of Allium sativum L.

Garlic and its bioactive compounds have also been shown to exhibit anti-inflammatory properties

(Morihara et al., 2017). In a study, the ethyl linoleate in garlic reduced the production of nitric

oxide (NO) and prostaglandin E-2 by down-regulating the expression of inducible NO synthase

(iNOS) and cyclooxygenase-2 (COX2) in lipopolysaccharide-stimulated RAW 264.7

macrophages (Park et al., 2014). Another study revealed that the garlic 14-kDa protein inhibited

the inflammatory mediators including NO, TNF-α, and interleukin (IL) -1β by inhibiting the

transcription factor nuclear factor-kappa B (NF-κB) signaling pathway in lipopolysaccharide-

stimulated J774A.1 macrophages (Rabe et al., 2015). In addition, AGE inhibited inflammation

in apolipoprotein E-knockout mice. The treatment of AGE reduced the level of tumor necrosis

factor-α (TNF-α) and the IL-1 receptor-associated kinase 4 and enhanced the activity of the

adenosine monophosphate-activated protein kinase (AMPK) in the liver ( Morihara et al., 2017).

Moreover, allicin could be used as a potential complementary treatment against the inflammatory

response induced by schistosome infection in BALB/c mice (Metwally et al., 2018).

Furthermore, garlic supplements alleviated osteoarthritis in obese or overweight patients by

reducing resistin (Dehghani et al., 2018).

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2.2 Bryophylum pinnatum lam

Bryophyllum pinnatum (lam.) Oken ( figure 2.4) plant is an environmental weed from the family

Crassulaceae ( Table 2.2), but commonly used traditionally as a medicine in different regions of

India mainly to treat urinary stones, as well as in other parts of world.

Bryophyllum pinnatum Lam. flourishes throughout the southern part of Nigeria and the one used

for this research is gotten from oja-jagun in Ogbomoso, Oyo State. It is well known for wound

healing properties. It also has considerable attention medicinal properties and folk medicine, as

well as in the contemporary medicine. The word meaning of Bryophyllum pinnatum: Derived

from Greek- Bryo means to sprout and phyllon is a leaf i.e. ability to propagate via leaf cutting,

pinnatum is from Latin feathered, winged.

Figure 2.4; Bryophylum pinnatum Lam

Bryophyllum pinnatum Lam, has many phytochemicals which includes flavonoids, saponins,

tannis and alkaloids (Narinderpal et al., 2014). Vitamins including ascorbic acid, riboflavin,

thiamine, niacin and minerals such as calcium, zinc and phosphorus are also present in the leaves

(Kanika 2011). The leaves have been reported to possess a variety of medicinal properties

including antimicrobial, antifungal, antiulcer, antihypertensive, antidiabetic, antiinflammatory,

14
analgesic and wound healing. Reports also show that the leaves possess anti-tumour, it has

sedative and muscle relaxant effect. It may also be effective in treatment of leishmaniasis (Mudi

et al., 2010). Herbal practitioners in Nigeria and other parts of Africa use the aqueous extract for

the treatment of coughs and as a prophylactic medicine for asthma. In Benin city, Nigeria, the

leaves are boiled filtered through a clean white cloth and the yield reconstituted for daily oral use

by asthmatic patients. The leaves and bark are bitter tonic, astringent to the bowels, analgesic,

carminative, useful in diarrhea and vomiting. It is applied externally and taken internally for all

types of pains and inflammations, various bacterial, viral and fungal infections, leishmaniasis,

earaches, upper respiratory infections, stomach ulcers, flu and fever . The herb is used to

facilitate the dropping of the placenta of newly born baby. Leaf juice is used in the treatment of

coughs, bronchial affections, blood dysentery, jaundice and gout (Anjo et al., 2010).. It is applied

externally and taken internally for all types of pains and inflammations, various bacterial, viral

and fungal infections, flu and fever (Mudi et al. 2010). The plant is commonly called a master

herb or cure for all antimalarial effect by a large community of herbal practitioners (Nayak et al.

2010). B. Pinnatum is rich in alkaloids, triterpenes, glycosides, flavonoids, cardienolides,

steroids, bufadienolides and lipids. The leaves contain a group of chemicals called

bufadienolides which are very active and possess antibacterial, antitumorous, cancer preventative

and insecticidal actions (Anjoo et al., 2010). Juice from the fresh leaves is usedfor the treatment

of jaundice in India because it was found be effective as evidenced by invivo and invitro

histopathological studies for hepatoprotective activityof plant and justifies the use of the juice of

the leaves in folk medicine for jaundice (Uchegbu et al., 2014). The plant has potent

antihistamine and anti- allergic activity.

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 Kingdom Plantae

Sub kingdom Tracheobiont

Division Spermatophyta

Subdivision Magnoliophyta

Class Magnoliopsida

Subclass Rosidae

Order Rosales

Family Crassulaceae

Genus Bryophyllum

Species Bryophyllum pinnatum

(Lam.)Oken

Table 2.2; Taxonomical Classification Bryophylum Pinnatum (Lam)

2.2.1 Botanical Description of Bryophyllum pinnatum (Lam)

Bryophyllum pinnatum (Lam) is succulent and grows between 1-1.5 m in height with bell-like

flowers, fleshy dark green leaves that are distinctively scalloped and trimmed in red and hollow

branched stems and usually fourangled (Pattewar, 2012). The leaves are 10-20 cm long,

opposite, and decussate, with the lower leaves being simple while the upper leaves are long-

petioled with 3-7 foliate. The leaf blade is 10-30 cm long, pinnately compound with 3-5 leaflets

which are oblong to elliptic and 6-8 x 3-5 cm (Khooshbu et al., 2019). Petiolules are 2-4 cm;

margin is crenate with rooting vegetative buds, which can develop into new plantlets (Khooshbu
16
et al., 2019). The plant flowers between November-March and the inflorescences are terminal,

10-40 cm long and the flowers are pendulous. The calyx is 2-4 cm and tubular; corolla is about 5

cm and reddish or purple in colour. The fruit which usually appears in April contains many seeds

that are ellipsoid, smooth, and striate (Nagaratna et al., 2015).

2.2.2. ETHNOMEDICINAL USES OF Bryophyllum pinnatum (Lam)

Different parts of B. pinnatum such as the leaves, bark, and juice, have found application in the

management of various diseases. The leaves have been used both internally as a tonic,

carminative, and as an astringent (Khooshbu et al.,2019). It has been used against many

bacterial, viral and fungi infections related diseases such as diarrhea and vomiting, upper

respiratory infections, and flu (Surendra et al., 2015). It has also been used as an analgesic and

anti-inflammatory when applied externally for several kinds of pain and inflammation. It has also

been used to treat fever, leishmaniasis , ulcer and hypertension (Rola et al.,2011). Other

medicinal properties of the leaves include its use as a disinfectant, hemostatic, emollient, and

refrigerant. Other reports highlight that the plant is useful in managing skin discolorations,

wounds, hemorrhoids, menorrhagia, boils, sloughing ulcers, ophthalmic disorders, burns, scalds,

corn, kidney stones, skin disorders, and edema (Anjo et al., 2010). The leaves have also been

used for treating edema of the legs, pulmonary infections, rheumatoid arthritis, cancer, boils,

epilepsy, hemorrhoids, piles, menorrhagia, skin discoloration, ophthalmia, menorrhagia and corn

(Rola et al.,2011). It has been used as anti-allergic, antihistamine, antifungal, and to increase

vascular integrity. The root infusion is used for treating epilepsy, while the juice is applied to

stop bleeding and enhance the healing of wounds. It is also used for managing headaches,

toothaches, earaches, eye infections, ulcers, boils, burns, and insect bites (Afzal et al., 2012). The

juice from the fresh leaves is used for treating smallpox, otitis, cough, asthma, palpitations,

17
headache, convulsion, and general debility. It is also used to treat coughs, bronchial infections,

blood dysentery, jaundice, and gout (Afzal et al., 2012). Given that B. pinnatum has several

applications in traditional medicine in different parts of the world, it is also called the “miracle

plant”. In the Southeastern part of Nigeria, it is used to ease the dropping of the placenta of a

newly born baby and for managing childhood illness (Surendra et al., 2015). The leaves are

included in common herbal remedies for several diseases such as hypertension, diabetes mellitus,

bruises, wounds, boils, abscesses, insect bites, arthritis, rheumatism, joint pains, headaches, and

body pains in many West African countries, including Nigeria. In Mexico, it is used to treat

menstrual problems and assist in childbirth (Kamboj et al.,2010). In Ecuador, the leaf infusion is

used for broken bones and internal bruises, and the Siona people apply the heated leaves

topically to boils and skin ulcers. In Peru, the leaf extract mixed with sugarcane rum is applied to

the temples for treating headaches while the overnight soaked leaves and stems are drunk for

fever, heartburn, urethritis, and respiratory conditions. An infusion of the root is also used for

managing epilepsy (Bum et al.,2011). In the West Indies, the Creoles use the leaf infusion to

treat fever and the roasted leaves for managing cancer and inflammations. In Brazil, the Palikur

are reported to use leaf juice mixed with coconut oil for migraines and headaches (Kamboj et

al.,2010).

2.2.3 PHYTOCHEMICAL CONSTITUENTS OF Bryophyllum pinnatum (Lam)

Different classes of bioactive compounds have been reported from B. pinnatum. Preliminary

phytochemical screening of different parts of the plant showed the presence of alkaloids,

phenols, flavonoids, anthocyanins, saponins, tannins, carotenoids, and coumarins (Anjo et al.,

2010). A metabolite profiling of the plant revealing different phytoconstituents has also been

18
reported (Pandey et al., 2020). Specific groups of phytochemicals from the plant are discussed

below.

[Link] Flavonoids

Several flavonoids have been identified from different parts of B. pinnatum (Furer et al., 2013).

Flavonoids from the leaves include flavones, flavans, flavanones, isoflavonoids, chalcones,

aurones and anthocyanidins. 5ʹ-Methyl-4ʹ,5,7- trihydroxyflavone and 4ʹ,3,5,7-tetrahydroxy5-

methyl-5ʹ-propanamide anthocyanidin were isolated by Okwu et al., to which the structure of the

latter appears biogenetically unlikey (Okwu et al., 2011). Quercitrin , a kaempferol diglycoside,

named kampinnatoside and identified as kaempferol 3-O-α-l-arabinopyranosyl-(1→2)- α-l-

rhamnopyranoside, flavonol and flavone glycosides quercetin-3-O-α-l-arabinopyranosyl (1→2)-

α-l-rhamnopyranoside and 4ʹ,5,7- trihydroxy-3ʹ,8-dimethoxyflavone 7-O-β-dglucopyranoside

with some degree of antileshmanial activity have been reported from the aqueous leaf extract

(Furer et al.2016). Seven kaempferol rhamnosides: kaempferitrin, kaempferol 3-O-(2-O-acetyl)-

α-L-rhamnopyranoside7-O-α-L-rhamnopyranoside, kaempferol 3-O-(2-O-acetyl)-α-L-

rhamnopyranosyl-7-Oα-L-rhamnopyranoside, kaempferol 3-O-(2- O-acetyl)-α-L-

rhamnopyranosyl-7-O-α-Lrhamnopyranoside, kaempferol 3-O-α-Dglucopyranosyl-7-O-α-L-

rhamnopyranoside, afzelin , and α-rhamnoisorobin were isolated from the ethyl acetate fraction

and reported with varying degree of antimicrobial activity (Tatsimo et al., 2012). Quercetin 3-O-

a-L-rhamnosyl-βD-xyloside , epigallocatechin-3-O-syringate , and luteolin were also reported

in the plant. These flavonoids are reported to be responsible for some pharmacological activities

such as antileishmanial, wound healing, antiinflammatory, and antioxidant properties (Furer et

al.,2016).

19
[Link] Phenanthrene derivatives

Two phenanthrene derivatives, 2-(9-decenyl)- phenanthrene and 2-(9-undecenyl)-phenanthrene

have been reported in the plant . A phenanthrene alkaloid identified as 1-ethanamino7-hex-1-

yne-5’-oxophenanthrene was also isolated from the ethanolic extract of the leaves. However, it

must be critically pointed out that the structure of the latter compound is rather unlikely from a

biogenetic perspective. These compounds contributed to the antimicrobial and antileishmanial

activities of the plant (Okwu et al., 2011).

[Link] Phenolcarboxylic and carboxylic acids

Phenolic compounds isolated from B. pinnatum include syringic acid, caffeic acid, malic acid,

gallic acid, isocitric acid, oxalic acid, succinic acid and threo-Dsisocitric acid (Anderson et al.,

2012) . Other phenolic compound reported in the plant are 4-hydroxy-3-methoxy-cinnamic acid,

4-hydroxybenzoic acid, p-hydroxycinnamic acid, (para-coumaric acid), protocatechuic acid,

while some carboxylic acids found in the plant are phosphoenolpyruvate, isocitric acid, succinic

acid, oxaloacetic acid . Phenols and phenylpropanoids are generally known to have antimicrobial

and antileishmanial activities (Tatsimo et al. ,2012).

[Link] Fatty acids

Fatty acids identified in B. pinnatum are palmitic acid, stearic acid, arachidic acid, and behenic

acid. Fatty acids from B. pinnatum were reported to show lymphocyte suppressive activity

(Pandey et al., 2020).

[Link] Steroids and phytosterols

Steroids and phytosterols are common secondary metabolites of plants. Several of this class of

compounds have been identified from different parts of B. pinnatum and linked to various

20
biological activities such cytotoxic and antibacterial effects (Cox - Georgian et al., 2019).. The

reported phytosterols include stigmasta-4,20,22-trienone, stigmasta-5-ene-3β-ol , (24S)-stigmast-

25- enol, stigmat-24-enol, bryophyllol, 25-methylstigmast-24-enol, clerosterol, 24-epiclerosterol,

β-sitosterol, 22-dihydrobrassicasterol, stigmasterol, campesterol, isofucosterol, codisterol,

clinesterol, peposterol, avenasterol, 25-methylergost-24-enol, and (24R)-Stigmasta-7,25-dienol,

and stigmat-5-en-3β-ol (Vadvalkar et al.,2014).

[Link] Bufadienolides

Bufadienolides are one of the main classes of active constituents in B. pinnatum (Roy, 2017).

These include bryotoxins A, B, and C which are very similar in their structure and possess

cytotoxic, antibacterial, and insecticidal effects (Faboro et al., 2016). The content of

bufadienolides in B. pinnatum was found to be higher in young leaves (Aldhebiani and Mufarah,

2017).. Other bufadienolides in the plant include bersaldegenin-1-acetate, bersaldegenin-3-

acetate, bersaldegenin-1,3,5-orthoacetate, bryotoxin A, and bryotoxin B. In other species,

additional bufadienolides such as daigredorigenin, diagremontianin, daigredorigenin-3-acetate,

kalantubosides A and B, and methyl daigremonate have been identified (Oufir et al., 2015).

[Link] Triterpenoids

Several studies reported the presence of triterpenoids in B. pinnatum (Kamjob et al., 2016).

Some of the terpenes in the species are Bryophollone, Bryophynol, Friedelin, Glut-5(6)-en-3β-

(ol, Glutinol, Pseudo taraxasterol, Taraxerol, Taxaxerone, α-Amyrin, α-Amyrin acetate, α-

Amyrin-β-D-glucopyranoside, β-Amyrin, β-Amyrin acetate , Ψ-Taraxasterol.

21
2.2.4. Pharmacological Activity of Bryophyllum pinnatum Lam

Several studies have reported the various pharmacological activities of Bryophyllum pinnatum to

include anti-ulcer, anti-diabetic, anti-inflammatory, antitumor, antioxidant, and

neuroprotective .These activities can be exploited for the development of potentially clinically

useful products and will be discussed in the following subsection.

[Link] Anti-inflammatory and Analgesic activity of Bryophyllum pinnatum lam.

Customarily, Bryophyllum pinnatum leaves and its flowers are used for the anti-inflammatory

and analgesic effects (Aprioku and Igbe, 2017). It contains flavonoids which have ability to

inhibit the cyclooxygenase enzyme and minimize the activity of α- tissue necrosis factor

(Ferreira et al., 2014). Leaves ethanolic extract was proved to be effective against the topical

acute and chronic inflammation which is due to cramming of the arachidonic acid pathway

(Chibli et al., 2014).

The study revealed the presence of Anti-inflammatory activity of leaf extracts of B. pinnatum

(pet-ether, chloroform, acetone, methanol, aqueous, alkaloidal fraction, flavonoids fraction,

phenol and phenolic acid, alkaloidal anhydride) in the doses of 500mg/kg each orally once a day

for a period of two days in Formaldehyde-induced hind paw edema in rats (Nagaratna et al.,

2015). Methanolic fraction showed also more or less significant inhibition of formaldehyde

induced edema in early phases while significant inhibition at later phases when compared to the

standard drug Indomethacin (Gupta et al., 2014)

22
[Link] Anti-allergy activity of Bryophyllum pinnatum lam.

An in vitro study has shown that Bryophyllum pinnatum is helpful in reducing allergy. Its anti-

allergic effect is due to the halting of antigen induced mast cell degranulation and also by

minimizing the secretion of histamine (Awuchi et al.,2020)

[Link] Anti-cancer activity of Bryophyllum pinnatum lam.

Bryophyllum pinnatum chloroform extract and its fractions have exhibited a concentration

dependent inhibition of human cervical cancer cell growth (Afzal et al., 2012). The fraction was

more potent than the extract and strong activity was observed against human papillomavirus

(HPV) which performs a vital job in the growth of cervical cancer (Mahata et al., 2012). From

the leaves, five bufadienolides have been separated and investigated for their inhibitory effects

on Epstein-Barr virus early antigen (Latif et al., 2019). From all the bufadienolides, an obvious

inhibition was exhibited by bryophyllin. Study outcomes have strongly recommended that the

Bryophyllum pinnatum bufadienolides could be the potential chemotherapeutic candidates to

treat the cancer (Afzal et al., 2012).

The in vitro study of B. pinnatum leaves crude extracts & its specific chromatographic fractions

revealed the presence of growth inhibitory activity in the human cervical cancer cells. The leaf

extract & its fraction F4 (Petroleum Ether: Ethyl Acetate: 50:50) have a dose dependent

cytotoxic activity, similarly the inhibition of viral transcription of HPV18 was lesser in cells

treated with fraction F4 which indicate higher concentration of active principles (Mahata et al.,

2014)

23
[Link] Anti-diabetic activity of Bryophyllum pinnatum lam.

For many years, Bryophyllum pinnatum has been utilized for its anti-hyperglycemic effects. The

aqueous extract of leaves, after postprandial and streptozotocin induced diabetes in rats has

exhibited striking hypoglycemic effects. Moreover, an advance investigation has confirmed its

effectiveness in heart diseases and in diabetes (Pattewar et al., 2012).

The study revealed the presence of anti-diabetic activity of B. pinnatum aqueous leaf extract in

four different doses (200, 400, 800mg/kg and 800mg/kg + glibenclamide 2mg/kg) in diabetic

induced rats (Glucose D- 3g/kg). The 200mg/kg aqueous extract resulted significant decrease in

the blood sugar level when compared with the other dose. But the mixture of 800mg/kg aqueous

extract + glibenclamide 2mg/kg showed more effective & efficient than the use of 200mg/kg and

the other single doses (Aransiola et al., 2014)

[Link] Antihypertensive activity of Bryophyllum pinnatum lam.

Bryophyllum pinnatum is used to treat various cardiovascular related disorders in folklore

therapeutics. Now it is confirmed that aqueous extract of the leaves has an antihypertensive

effect on rats which justify its use in folk medicines. It has been demonstrated that the extract has

potent anti- oxidant effect on aorta thus plays a significant role in the lessening of blood pressure

(Bopda et al., 2014).

The study has shown the presence of Antihypertensive activity of B. pinnatum aqueous &

methanolic leaf extracts (50-800mg/kg i.v. or i.p.) on arterial blood pressure & heart rates of

normotensive & spontaneously hypertensive rats (Nagaratna et al., 2015). The hypotensive effect

was more pronounced in the hypertensive than in normotensive rats. Even the leaf extracts (0.25-

24
5.0 mg/ml) also produced dose-dependent decrease in the rate & force of contractions of guinea-

pig isolated atria (John et al., 2014)

[Link] Anti leishmanial activity of Bryophyllum pinnatum lam.

Flavonoids present in Bryophyllum pinnatum are responsible for its anti leishmanial effects. In

the aqueous extract of leaves of Bryophyllum pinnatum. Chemically defined natural substances

showed antileishmanial activity in Bryophyllum pinnatum extracts(coumarin and quercetin). B.

pinnatum has antileishmanial flavonoid quercitrin. A function in antileishmanial activity oral

administration of these flavonoids proved more efficient against leishmaniasis in mice than

intravenous. To prevent leishmaniasis, plants may stimulate reactive nitrogen intermediates in

macrophages (Kumar et al., 2021)The quercetin aglycone type structure and a rhamnosyl unit

linked at C-3 were found to be essential for anti leishmanial activity (Mahata et al., 2012).

[Link] Urolithic activity of Bryophyllum pinnatum lam.

Bryophyllum pinnatum is used for the treatment of renal stones in traditional medicines. Leaves

aqueous extract markedly decreases the level of urine oxalate and consequently it can be helpful

in the cure of urolithiasis (Shukla et al., 2014). Bryophyllum pinnatum has been proven

beneficial in the reduction of renal stones because it enhances the excretion of oxalate crystals by

reducing the size of crystals and by converting them from dehydrate crystals to calcium oxalate

monohydrate form (Yasir and Waqar, 2011).

[Link] Gastroprotective/ Anti-ulcer activity of Bryophyllum pinnatum lam.

Bryophyllum pinnatum possess gastroprotective effects and it has been verified by its striking

dose dependent defensive effect onS ethanol induced gastric injury (Sharma et al., 2014).

25
However, further studies should be carried out to validate its use in gastric ulcers (Sharma et al.,

2014).

[Link] Hepatoprotective activity of Bryophyllum pinnatum lam.

Bryophyllum pinnatum has been monitored for its hepatoprotective activity. In rats, carbon

tetrachloride stimulated hepatic injury was induced and found that the ethanolic extract of the

leaves reduces the levels of liver enzymes, serum bilirubin, serum cholesterol and serum total

protein. Results have illustrated that the herb has an obvious hepatoprotective activity. Increased

regeneration of hepatocytes and microsomal enzymes inhibition also defend the liver from

damage (Sharma et al., 2014).

2.2.4. 10 Anti-oxidant activity of Bryophyllum pinnatum lam.

Bryophyllum pinnatum is tested for its anti-oxidant activity by metal chelating assay, 1,1-

diphenyl-2-picrylhydrazyl (DPPH) assay and 2,2’- azinobis-(3-ethylbenzothiazoline-6-sulfonic

acid) (ABTS) assay. Study outcomes have indicated that the ethanolic extract has marked anti-

oxidant activity (Sindhu and Manorama, 2015). Roots extracts have also exhibited the anti-

oxidant effects when analyzed by DPPH assay (Gupta and Banerjee, 2011).

[Link] Nephroprotective effects of Bryophyllum pinnatum lam.

Bryophyllum pinnatum is widely used for its nephroprotective activity in folklore and the

rationale behind its use has been proven by the studies. Results of an investigation have shown

that this effect is dose dependent. The nephroprotective activity against the genatmicin induced

nephrotoxicity on the Wistar rat kidney was tested and it is anticipated that this effect is due to

the plant anti-oxidant and radical scavenging properties. (Schuler et al., 2012).

26
2.2.4 .12 Neurosedative and muscle relaxant activity of Bryophyllum pinnatum lam.

Bryophyllum pinnatum has marked effect on the CNS and it has been proven that the methanolic

extract produced a significant change in behavior pattern. A study results have demonstrated that

the herb caused the CNS depression and dose-dependent stimulation of pentobarbitone sleeping

time. Another study has also suggested that it is useful in treating the sleep troubles during

pregnancy (Afzal et al., 2013). The medicinal plant is helpful for the treatment and management

of seizures and that was confirmed by testing on mice. It showed a dose dependent increase onset

and duration of pentobarbitone-induced sleep and decline of exploratory activities in the head-

dip and evasion tests. A dose-dependent muscle incoordination has been verified in the inclined

screen, traction and climbing tests. In both strychnine and picrotoxin induced seizures it caused a

late onset of convulsions (Quazi et al., 2011).

[Link] Uterine relaxant activity of Bryophyllum pinnatum lam.

In traditional therapeutics, the plant is used for tocolysis and the rationale behind its use has

proven by in vitro studies and further research is still required. The effect of leaf press juice and

its chemical fractions were studied on human myometrial strips and were found to be useful in

relaxing the myometrial strips (Wächter et al., 2011).

2.3 LI VER

The liver is a critical organ in the human body that is responsible for an array of functions that

help support metabolism, immunity, digestion, detoxification, vitamin storage among other

functions. It comprises around 2% of an adult's body weight. The liver is a unique organ due to

27
its dual blood supply from the portal vein (approximately 75%) and the hepatic artery

(approximately 25%) (Abdel-Misih et al.,2010).

The functional unit of the liver is the lobule. Each lobule is hexagonal and a portal triad

(portal vein, hepatic artery, bile duct) sits at each corner of the hexagon. The foundation of the

lobule is composed of hepatocytes, which have physiologically distinct apical and basolateral

membranes (Si-Tayeb et al.,2010).

2.3.1 Functions of Liver

Bile is a vital physiological fluid crucial in eliminating poisons the kidneys cannot excrete.

Furthermore, it enhances the assimilation and breakdown of lipids via the excretion of bile salts

and acids. Hepatocytes are accountable for synthesizing bile, a multifaceted fluid primarily

consisting of electrolytes, water, bile acids, bile pigment, bile salts, bilirubin, cholesterol, and

phospholipids, among other components (Noussios et al., 2017). Hepatocytes secrete bile into

the bile duct, traversing via a network of channels of varying widths before reaching the

duodenum or being deposited in the gallbladder for accumulation. The precise location is

dictated by the pressures exerted by the duct and the sphincter of Oddi. Upon being released into

the duodenum, bile undergoes circulation inside the enterohepatic system, which carries out its

physiological role within the intestines (Carneiro et al., 2019). The constituents of bile that are

not excreted undergo a process of recycling facilitated by intestinal bacteria, leading to their

conversion into bile acids. Subsequently, the bile acids are absorbed within the ileum and

transported back to the liver (Kalra et al., 2023).

The liver serves as the site for storing or metabolizing fat soluble vitamins. The alpha- and

gamma-tocopherol forms are how Vitamin E is transported to the liver. Despite the liver’s

28
inability to store or metabolize vitamin K, it remains an essential component for the liver enzyme

gamma-glutamyl carboxylase (Kalra et al., 2023).

The liver is involved in metabolic detoxification, transforming both endogenous and exogenous

substances, including medications and hormones, into metabolites that are less biologically

active and less harmful. These metabolites are then eliminated from the body via the bowels or

the kidneys. The liver plays a crucial part in detoxification concerning alcohol, amphetamines,

hormones, steroids, and barbiturates (Carneiro et al., 2019). Its primary function is to prevent the

excessive buildup of these substances and mitigate any potential negative effects. While

metabolic detoxification is typically beneficial, there are instances where it can adversely affect

hepatocytes. One illustration of the detrimental effects of alcohol abuse is the potential harm it

can inflict upon hepatocytes, primarily due to the metabolic byproducts of alcohol, including

acetaldehyde and hydrogen. The metabolic byproducts elicit an augmentation in adipose tissue

deposition, which has the potential to impair hepatic functionality (Ozougwu 2017).

Hepatocytes produce enzymes involved in detoxification, which can be categorized into phases:

phase I and II. Phase I enzymes function by introducing hydrophilic polar groups, such as

hydroxyl (-OH), to lipophilic molecules, thereby enhancing their hydrophilicity. In the interim,

phase II enzymes conjugate hydrophilic components (e.g., sugar or peptide, such as glutathione)

to the polar moieties introduced by phase I enzymes. The liver may be susceptible to harm if

there is a disruption in the activity of phase II enzymes, leading to an accumulation of reactive

chemicals generated during phase I metabolism (O’Brien et al., 2015).

The process of heme oxidation predominantly occurs within the hepatic tissue. Unconjugated

bilirubin is generated through the conversion of heme to biliverdin. Most of it is introduced into

the bile and subsequently eliminated through the excretion of bile in fecal matter. A fraction of

29
the substance dissolves within the bloodstream after filtration before renal excretion (O’Briena et

al., 2015).

The process of deiodination, specifically the conversion of thyroxine (T4) to triiodothyronine

(T3), takes place inside the hepatic tissue, hence playing a significant role in the overall

functionality of thyroid hormones (Kalra et al., 2023). The liver is responsible for synthesizing

most plasma proteins found in the human body, including albumin, protein C, binding globulins,

protein C, protein S, and all clotting factors except for factor VIII, which are created through

both the intrinsic and extrinsic pathways (Carneiro et al., 2019).

The liver possesses a substantial capacity to retain a significant volume of blood due to its

abundant presence of blood arteries. In hemorrhage, the liver can secrete blood to sustain the

overall blood volume inside the circulatory system. Due to its anatomical connection with the

gastrointestinal tract, the liver is prone to infiltrating germs or foreign particles introduced into

the portal vein system. Kupffer cells can eradicate bacteria and serve as a protective barrier

against infection (Ozougwu 2017).

2.3.2 Anatomical description of Liver

Humans have the largest smooth-surfaced organ, the liver. The liver makes up about 2-3% of the

total body weight in humans. Men’s livers typically weigh 1800 grams, while women’s livers

weigh only 1400 grams (Sibulesky, 2013). Located under the right hemidiaphragm in the right

upper quadrant of the abdomen, costae surround and protect this organ (Juza et al.,2014). The

liver is completely encased in the Glisson capsule except for the exposed area next to the

diaphragm (Llewellyn et al., 2022). When viewed from the outside, the falciform ligament

separates the liver into a right and a left lobe (Sibulesky, 2013). The liver is joined to the anterior

30
abdominal wall by this ligament. The ligament teres, containing the umbilical vein’s final stages

of development, are located at the base of the liver (Mahadeva 2020). The diaphragm and

visceral surfaces of the liver can be considered its two surfaces, with the unmarked anterior,

lateral, superior, and posterior portions of the diaphragm surface (Mahadeva 2020).

The umbilical fissure and the falciform ligament separate the liver into two lobes, with the right

lobe more extensive than the left lobe (Sadler 2019). The caudate lobe and lobe quadratus

comprise the right lobe of the liver. The lobe quadratus and caudate lobe are divided by the

transverse hilar fissure, where the porta hepatis enters the liver. The gallbladder and umbilical

fissure both surround the lobe quadratus on either side (Kalra et al., 2023). Anterior to the hilar

fissure is found in the caudate lobe, sometimes called Spigel’s lobe. The liver contains two

primary and two accessory lobes, each divided by easily recognizable fissures (Sadler 2019).

The liver is divided into eight functional parts, each with its portal pedicles, according to

Couinaud’s classification (Figure4.5). The branches of the right and left hepatic arteries, the

hepatic portal vein, and the hepatic duct form the portal pedicle. The liver’s left lobe is divided

into segments II, III, and IV. V, VI, VII, and VIII are the segments that make up the right lobe

(Sibulesky, 2013). Segment I is in the caudate lobe, (Table4.1).

The right hepatic vein divides the right lobe of the liver into anterior and posterior portions. The

right and left lobes of the liver are separated by the intermediate hepatic vein, which connects to

the inferior vena cava. The left hepatic vein separates the medial and lateral parts of the left lobe.

The portal vein divides the liver into superior and inferior parts (Figure 2.5) (Abdel-Misih et al.,

2010).

Understanding these liver parts is crucial because they provide guidelines for doing liver

resection. According to the Couinaud classification, segmentectomy removes an anatomical

31
segment (Figure 2.5). Segments II and III are resected using the left lateral segmentectomy

technique, while segment IV is resected using the left medial segmentectomy technique.

Segments V and VIII undergo a right anterior segmentectomy, whereas segments VI and VII

have a right posterior segmentectomy (Kalra et al., 2023). Left hepatectomy is used when the

resection is done on segments II, III, and IV. Like removing segments V through VIII, right

hepatectomy is also performed. Segments V, VI, VII, and VIII that reach segments I, IV, or both

are removed during an extended right hepatectomy. While an extended left hepatectomy is when

segments I, V, VIII, or a mix of segments IV, II, and III are removed (Juza et al.,2014).

Figure 2.5; morphology of the liver (Abdel-Misih et al., 2010).

32
Liver lobes Liver segmentation Location

Left lobe II and III Lateral segment

IV Medial segment

Right lobe V and VIII Anterior segment

VI and VII Posterior segment

Caudate lobe I On posterior liver

Table 2.3; Liver segmentation

2.3.3 Liver Function Biomarker

[Link] Serum Bilirubin

Bilirubin is the catabolic product of haemoglobin produced within the reticuloendothelial system,

released in unconjugated form which enters into the liver, converted to conjugated forms

bilirubin mono and diglucuronides by the enzyme UDP-glucuronyltransferase (Iluz-Freundlich et

al., 2020). Normal serum total bilirubin varies from 2 to 21μmol/L. The indirect (unconjugated)

bilirubin level is less than 12μmol/L and direct (conjugated) bilirubin less than 8μmol/L (Ribeiro

et al., 2019) . The serum bilirubin levels more than 17μmol/L suggest liver diseases and levels

above 24μmol/L indicate abnormal laboratory liver tests (Vagvala et al., 2018). Jaundice occurs

when bilirubin becomes visible within the sclera, skin, and mucous membranes at a blood

concentration of around 40 μmol/L (Gupta et al.,2018) . The occurrence of unconjugated

hyperbilirubinemia due to over production of bilirubin, decreased hepatic uptake or conjugation

or both. It is observed in genetic defect of UDP-glucuronyltransferase causing Gilbert\'s

syndrome, Crigler-Najjar syndrome and reabsorption of large hematomas and ineffective

erythropoiesis (Kwo et al., 2017). In viral hepatitis, hepatocellular damage, toxic or ischemic

liver injury higher levels of serum conjugated bilirubin is seen. Hyperbilirubinemia in acute viral

33
hepatitis is directly proportional to the degree of histological injury of hepatocytes and the longer

course of the disease (Vagvala et al., 2018). It has been observed that the decrease of conjugated

serum bilirubin is a bimodal fashion when the biliary obstruction is resolved (Leoni et al., 2018

). Parenchymal liver diseases or incomplete extrahepatic obstruction due to biliary canaliculi

give lower serum bilirubin value than those occur with malignant obstruction of common bile

duct but the level remains normal in infiltrative diseases like tumours and granuloma(Hustead et

al., 2017 ). Raised Serum bilirubin from 20.52 μmol/L to 143.64μmol/L in acute inflammation of

appendix has been observed (Prati et al.,2010). In normal asymptomatic pregnant women total

and free bilirubin concentrations were significantly lower during all three trimesters and a

decreased conjugated bilirubin was observed in the second and third trimesters (Kwo et al.,

2017). The recent study has shown that a high serum total bilirubin level may protect neurologic

damage due to stroke (Vagvala et al., 2018).

[Link] Alanine amino transferase (ALT)

ALT is found in kidney, heart, muscle and greater concentration in liver compared with other

tissues of the body. ALT is purely cytoplasmic catalysing the transamination reaction (Ruhl et

al.,2010) . Normal serum ALT is 7–56 U/ L (Prati et al.,2010). Any type of liver cell injury can

reasonably increases ALT levels. Elevated values up to 300 U/L are considered nonspecific.

Marked elevations of ALT levels greater than 500 U/L observed most often in persons with

diseases that affect primarily hepatocytes such as viral hepatitis, ischemic liver injury (shock

liver) and toxin-induced liver damage. Despite the association between greatly elevated ALT

levels and its specificity to hepatocellular diseases, the absolute peak of the ALT elevation does

not correlate with the extent of liver cell damage (Coates 2011). Viral hepatitis like A, B, C, D

and E may be responsible for a marked increase in aminotransferase levels. The increase in ALT

34
associated with hepatitis C infection tends to be more than that associated with hepatitis A or B

(Ruhl et al.,2010) . Moreover in patients with acute hepatitis C serum ALT is measured

periodically for about 1 to 2 years (Ribeiro et al., 2019). Persistence of elevated ALT for more

than six months after an occurrence of acute hepatitis is used in the diagnosis of chronic

hepatitis. Elevation in ALT levels are greater in persons with nonalcoholic steatohepatitis than in

those with uncomplicated hepatic steatosis (Manne et al., 2018). In a recent study the hepatic fat

accumulation in childhood obesity and nonalcoholic fatty liver disease causes serum ALT

elevation. Moreover increased ALT level was associated with reduced insulin sensitivity,

adiponectin and glucose tolerance as well as increased free fatty acids and triglycerides (Leoni et

al., 2018 ). Presence of Bright liver and elevated plasma ALT level was independently

associated with increased risk of the metabolic syndrome in adults (Koenig et al., 2015) . . ALT

level is normally elevated during 2nd trimester in asymptomatic normal pregnancy]. In one of

the study, serum ALT levels in symptomatic pregnant patients such as in hyperemesis

gravidarum was 103.5U/L, in pre-eclampsia patients was 115U/L and in haemolysis with low

platelet count patients showed 149U/L (Coates 2011). However in the same study ALT rapidly

drops more than 50% of the elevated values within 3 days indicating the improvement during

postpartum (Rozga et al., 2013). One of the recent study has shown that coffee and caffeine

consumption reduces the risk of elevated serum ALT activity in excessive alcohol consumption,

viral hepatitis, iron overload, overweight, and impaired glucose metabolism (Leoni et al., 2018 ).

[Link] Aspartate amino transferase (AST)

AST exist two different isoenzyme forms which are genetically distinct, the mitochondrial and

cytoplasmic form. AST is found in highest concentration in heart compared with other tissues of

the body such as liver, skeletal muscle and kidney. Normal serum AST is 0 to 35U/L (Ribeiro et

35
al., 2019). Elevated mitochondrial AST seen in extensive tissue necrosis during myocardial

infarction and also in chronic liver diseases like liver tissue degeneration and necrosis (Scarà et

al., 2015). About 80% of AST activity of the liver is contributed by the mitochondrial

isoenzyme, whereas most of the circulating AST activity in normal people is derived from the

cytosolic isoenzyme (Kell et al., 2014). However the ratio of mitochondrial AST to total AST

activity has diagnostic importance in identifying the liver cell necrotic type condition and

alcoholic hepatitis (Lippi et al., 2019). AST elevations often predominate in patients with

cirrhosis and even in liver diseases that typically have an increased ALT (Cheong et al., 2011).

[Link] Alkaline phosphatase (ALP)

ALP is present in mucosal epithelia of small intestine, proximal convoluted tubule of kidney,

bone, liver and placenta. It performs lipid transportation in the intestine and calcification in bone.

The serum ALP activity is mainly from the liver with 50% contributed by bone (Sharma et al.,

2014). Normal serum ALP is 41 to 133U/L (Sharma et al., 2014). In acute viral hepatitis, ALP

usually remains normal or moderately increased. Elevation of ALP with prolonged itching is

related with Hepatitis A presenting cholestasis. Tumours secrete ALP into plasma and there are

tumour specific isoenzymes such as Regan, Nagao and Kasahara (Verma et al., 2012). Hepatic

and bony metastasis can also cause elevated levels of ALP. Other diseases like infiltrative liver

diseases, abscesses, granulomatous liver disease and amyloidosis may cause a rise in ALP.

Mildly elevated levels of ALP may be seen in cirrhosis, hepatitis and congestive cardiac failure

(Verma et al., 2012). Low levels of ALP occur in hypothyroidism, pernicious anaemia, zinc

deficiency and congenital hypophosphatasia (Schilsky et al., 2017). ALP has been found

elevated in peripheral arterial disease, independent of other traditional cardiovascular risk factors

(Keerl et al., 2020). Often clinicians are more confused in differentiating liver diseases and bony

36
disorders when they see elevated ALP levels and in such situations measurement of gamma

glutamyl transferase assists as it is raised only in cholestatic disorders and not in bone diseases

(Koenig et al., 2015).

37
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Collection and Preparation of Plants Extracts

Bryophyllum pinnatum Lam. and Allium sativum L. these plants were collected in Oja –

jagun, Ogbomoso town, Oyo state and were authenticated by Prof. A,T.J Ogunkunle a

taxonomist from the Department of Pure and Applied Biology, Ladoke Akintola University of

Technology, Ogbomoso, Oyo State, Nigeria. The voucher numbers of the plants were deposited

at the University Herbarium which is LHO754 and LHO756 respectively. The plants material

was pulverized into powder with a kitchen blender.

3.1.2 Preparation of Methanol Extract of Collected Plants Materials

Five hundred (500 g) of the pulverized dried plant material was soaked in 1.5 L of analytical

grade methanol, and was kept in dark cupboard at room temperature for 72 hours. On the 3 rd day,

the mixture (solvent and powdered plant materials) was filtered with a muslin cloth and

subsequently with filter paper. The filtrate was concentrated under pressure at 50 ◦C with a

rotatory evaporator (Senco vaccuma evaporator). The dried crude extract obtained was stored in

airtight dark bottle and kept in the refrigerator (Haier Thermocool, Shandong, China) until

further use.

3.2 Acute Toxicity of the Plants Extract

Evaluation of acute and lethal dosage (LD50) of the plant combined extract of 2:1 was determined

according to the method of Lorke (1983).

3.3 Experimental Animals

Twenty-eight (28) healthy male Wistar rats weighing 100–120 g were obtained from a

commercial breeder in Ogbomoso, Oyo state. They were randomly separated into 4 groups of 7

38
animals; the animals were fed with normal rat feed and given combined extract orally using

cannula.

3.4 Dose of extract

Twenty –eight (28) rats were separated into four (4) groups

 Group A: 500mg/kg of the extract for 21days

 Group B: 250mg/kg of the extract for 21 days

 Group C: 167mg/kg of the extract for 21days

 Group D (control): water for 21days

3.5 Animal Sacrifice and Isolation of Organs

After 21 days, the experimental animals were allowed to fast for 12 hours before the

sacrifice was conducted. The experimental rats were sacrificed through cervical dislocation.

Blood was collected via cardiac puncture, while the liver were isolated for various biochemical

analyses and stored in freezer at -4℃.

3.5.1 Collection of Blood Serum

The collected blood was collected through cardiac puncture using a 5 mL syringe and

transferred into plain sample bottles. The blood samples were centrifuged at 4000 rpm for 10 min

to obtain the serum which was stored in the freezer at -180C.

3.6 Determination of Liver Function Markers

3.6.1 Determination of Aspartate Amino Transferase (AST) Activity

The Aspartate amino transferase (AST) activity in the plasma of experimental rats was assayed

according to Reitman and Frankel (1957)

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[Link] Principle

AST
α- ketoglutarate + L-aspartate L-glutamate + oxaloacetate

NADH oxidises oxaloacetate to L-malate by malate dehydrogenase (MDH)

+ MDH +
Oxaloacetate +NADH + H L- malate + NAD

The rate of decrease in NADH is spectrophotometrically measured and is directly proportional to

the activity of AST enzyme.

[Link] Procedure

The sample mixture contained 0.1 ml of sample and 0.5 ml of reagent R1a (DL-Aspartate and α-

ketoglutarate), while the reagent sample contained 0.1 ml distilled water and 0.5 ml of reagent

R1a. The solutions were mixed and incubated for 30mins at 37°C. There was addition of 0.5ml

of solution R2 (2, 4-Dinitrophenylhydrazine) with mixing. The mixture was allowed to stand for

20mins at about 25°C. Sodium hydroxide (5.0 ml, 0.4 M) was added and absorbance was read

spectrophotometrically at 546nm after 5mins.

3.6.2 Assay of Alanine Amino Transferase (AST) Activity

The alanine amino transferase (ALT) activity in the serum of the experimental rats was assayed

according to Reitman and Frankel (1957)

[Link] Principle:

ALT
α- ketoglutarate + L-alanine L-glutamate + pyruvate

NADH oxidises pyruvate to L- lactate by lactate dehydrogenase (LDH)

+ LDH
Pyruvate +NADH + H L- lactate + NAD

40
The rate of decrease in NADH is spectrophotometrically measured and is directly proportional to

the activity of ALT enzyme.

[Link] Procedure

The sample mixture contained 0.1 ml of sample and 0.5 ml of reagent R1b (DL-Alanine and α-

ketoglutarate), while the reagent sample contained 0.1 ml distilled water and 0.5 ml of reagent

R1b. The solutions were mixed and incubated for 30mins at 37°C. There was addition of 0.5 ml

of solution R2 (2, 4-Dinitrophenylhydrazine) with mixing. The mixture was allowed to stand for

20mins at about 25°C. Sodium hydroxide (5.0 ml, 0.4 M) was added and absorbance was read

spectrophotometrically at 546nm after 5mins.

3.6.3 Determination of Alkaline Phosphatase Activity

[Link] Principle

Alkaline phosphatase (ALP) catalyses the hydrolysis of p-nitophenyl phosphate at pH 10.4, thus

liberating p-nitrophenol and phosphate respctively. The rate of p-nitrophenol formation

measured photometrically, is proportional to the catalytic concentration of alkaline phosphate

present in the sample.

ALP
p-Nitrophenylphosphate + H2O p-Nitrophenol + Phosphate.

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3.6.3.2Procedure

Pipette into a clean test tube 1.2 ml of working reagent (WR) and add 20 μL of sample, mix and

incubate for 1 minute at 37 0C. Pour the mixture into a clean match cuvette (1.0cm light path) and

read read the absorbance on a spectrophotometer at 405 nm at 1 minute interval, thereafter for 3

minutes.

3.6.4 Determination of Total Bilirubin Concentration

[Link] Principle

Bilirubin is converted to colored azobilirubin by diazotized sulphanilic acid. Bilirubin

glucuronide reacts directly in aqueous solution (Bilirubin direct), while free bilirubin (plasma-

bound) is first solubilized with Dimethylsulphoxide (DMSO) to react (Bilirubin indirect). In the

determination of the indirect bilirubin, the direct is also determined and the result corresponds to

total bilirubin. The intensity of the colour formed is proportional to the bilirubin concentration.

[Link] Procedure

The reaction mixture for Total bilirubin contained 1.5ml of reagent R2, 50µl of R3 and 100µl of

sample, while the rection mixure for its blank contained 1.5 ml of R2 and 100 µl of calibrator.

The reaction mixture for direct bilirubin contained 1.5ml of reagent R1, 50µl of R3 and 100µl of

sample, while that of its blank contained 1.5ml of R1 and 100µl of calibrator. The mixtures was

incubated for 5 minutes at about 20 0C, and the absorbance was read spectrophotometrically at

555nm.

42
43
 CHAPTER FOUR

4.0 Results

4.1 Biomarkers Activity

4.1.1 Effect of combined extract on Liver ALT Concentration.

The ALT concentration in male wister rats administered with methanol extract shown in

figure 4.1 shows that there is a significant difference (p <0.05) in 500mg/kg when compared to

control and there is an increase in ALT concentration in 500mg/kg compared to control.

250mg/kg and 167mg/kg show no significant difference (p>0.05) when compared to control. The

extract shows the effect of dose dependent.

Figure4.1;Effect of Combined extract on Liver ALT Concentration.

44
4.1.2 Effect of Combined extract on Liver AST Concentration.

The AST concentration in male wister rats administered with methanol extract shown in

figure 4.2 shows that there is a significant difference (p <0.05) in 500mg/kg when compared to

control and there is an increase in ALT concentration in 500mg/kg compared to control.

250mg/kg and 167mg/kg show no significant difference (p>0.05) when compared to control.

Dose dependent effect was shown.

Figure4.2;Effect of Combined extract on Liver AST Concentration.

45
4.1.3 Effect of Combined extract on Liver ALP Concentration.

The ALP concentration in male wister rats administered with methanol extract shown in

figure 4.3 shows that there is no significant difference (p>0.05) in 500mg/kg, 250mg/kg and

167mg/kg when compared to control. The effect of the extract is dose independent

Figure4.3;Effect of combined extract on Liver ALP Concentration.

46
4.1.4 Effect of Combined extract on Liver Total billirubin Concentration.

The total bilirubin concentration in male wister rats administered with methanol

combined extract shown in figure 4.4 shows that there is no significant difference in 500mg/kg,

250mg/kg and 167mg/kg (p>0.05) when compared to control. From the graph, it was shown that

the extract effect is dose independent.

Figure 4.4; Effect of Combined extract on Liver Total bilirubin Concentration.

47
4.1.5 Effect of Combined extract on Liver Conjugated Bilirubin Concentration.

The conjugated bilirubin concentration in male wister rats administered with methanol

extract shown in figure 4.5 shows that there is no significant difference (p>0.05) in 500mg/kg,

250mg/kg and 167mg/kg when compared to control. The effect of the erxtract is dose

independent.

Figure 4.5; Effect of Combined extract on Liver Conjugated Bilirubin.

48
4.1.6 Effect of Combined extract on Liver Weight

The liver weight in male wistar rats administered with combined methanol extract of

[Link] and A. sativum shown in figure 4.6 shows that there is no significant difference

and its dose dependent such that the lower the dose the increase in the liver weight.

4.6; Effect of combined extract on the liver weight.

49
 CHAPTER FIVE

DISCUSSION

The liver is a prime target organ of any form of toxicity, this is because various studies

corroborate the view that the liver plays a critical role in the biotransformation of chemical

substances and facilitates their elimination from the body [Phang-Lyn et al., 2023) Assessment

of liver damage was determined by serum concentrations of ALT, AST and ALP. These liver

transaminases AST and ALT provide information about the state and integrity of the liver

(O’Shea et al.,2010). Although serum levels of AST and ALP are measured clinically as bio

markers for liver health, an increase in ALT activity is more pronounced (Lala et al., 2023).

When body tissues or an organ such as liver is damaged, variety of enzymes usually found in the

cytosol are released into the bloodstream causing the level of the enzymes to rise. The amount of

these enzymes in the blood is directly related to the extent of damage to the liver” (Lala et al.,

2023).

According to Omoniyi et al., (2020) in a studies that was carried out on toxicological and

reversibility of Bryophyllum pinnatum, it was shown that there is significant increase in the

concenraion of ALP, AST and ALT which shows that garlic is toxic to the liver but there is no

significant decrease in bilirubin although at high dosage 1000mg/kg.

According to Hauzaifa et al., (2013), it was shown that significant increases (P<0.05) in

the activities of ALT, AST, ALP and in the levels of total bilirubin in a dosage of 400-

550mg/kg.

The result for this studies shows that there is significant increase in the concentration of

ASTand ALT and from some studies that has been carried out on the toxic effect of the Allium

50
sativum and [Link] separately shows that there are significant increase in the biomarkers

concentration.

51
 CONCLUSION

Bryophyllum pinnatum (Lam) and Allium sativum L. is a medicinal plant that has been widely

used in r treating several diseases such as wound healing, antiulcer, antidiabetic,

anti-inflammatory, antinociceptive, and antibacterial activity, the list of following chemical

constituents flavonoid, alkaloid, saponin, and triterpenoid, allin, allicin e.t.c are responsible for

this activity. With their amazing usefulness for treating some diseases, their toxic effect should

be also considered. This review shows the toxic effect of the exacts on liver function test and has

been showed that they have toxic effect on ALT and AST. Although, more research should be

carried out.

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