CHAPTER ONE

INTRODUCTION

1.0 BACKGROUND OF THE STUDY

means a lustrous bitter substance, and ‘vera’, word which means ‘true’ in Latin. The Aloe

vera plant belongs to Liliaceae family and is considered one of the oldest and most

commonly used medicinal plants. It was historically used as a laxative or acids (Misir et al.,

2014).

Aloe vera has been used for centuries in health, beauty, medicine, and skin care (Gupta et al.,

2012). Other therapeutic benefits of aloe vera include treating teeth and gums, treating

constipation, antioxidant and antibacterial properties, and protecting against radiation (Maan

et al., 2018). Aloe vera is also used commercially in yogurt, beverages, and some desserts

(Mukherjee et al., 2013). Today, Aloe vera is used as a traditional remedy for a variety of

conditions and is found in dietary supplements and food products. Aloe vera gel can also be

found in different skin products, including lotions and sun blocks (NCCAM, 2012).

Aloe vera is praised for its healing properties, but the scientific bases for these actions have

not been adequately ascertained. Therefore, this study was conducted to fill the above

knowledge gap by performing a phytochemical analysis of the plant to identify and quantify

its phytochemical constituents. This may be helpful for researchers in the field to identify the

potential health and cosmetic benefits of aloe vera as well as its potential benefits in the food

industries.

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Aloe barbadensis Miller plant shows a high-water content, ranging from 99% to 99.5%, while

the 0.5– 1.0% solid material contains over 200 different potentially active compounds,

including enzymes, minerals, vitamins, simple and complex polysaccharides, phenolic

compounds, and organic.

The chemistry of the aloe vera has been studied for many years. Although for the analysis of

the phytochemicals in aloe vera, different methods such as fluoro-photometry, Gas

chromatography – mass spectrometry (GC-MS), thin layer chromatography, size exclusion

chromatography, High-performance liquid chromatography (HPLC), Liquid

chromatography–mass spectrometry (LC/MS), atomic-absorption spectrometry, counter

current chromatography, capillary electrophoresis and micellar electrokinetic chromatography

have been adopted; the GC-MS (Salisu and Shema, 2020) and HPLC (Mukherjee et al.,

2013) methods has been widely applied to analyse the components in aloe vera. Chemical

analysis reveals that Aloe vera contains various carbohydrate polymers, notably

glucomannans, along with a range of other organic and inorganic components (Mukherjee et

al., 2013). Although aloe vera is not well known when its medical applications were first

discovered, it has a long association with folkloric application. Three preparations of the aloe

vera used mainly in medicine are quite different in their chemical composition and

therapeutic properties, aloe vera latex; aloe vera gel; and aloe whole leaf (aloe extract). Aloe

vera latex is used for its laxative effect; aloe vera gel is used for skin ailments, such as wound

healing, psoriasis, genital herpes, and by oral administration in diabetics and hyperlipidaemic

2
patients and to heal gastric ulcers; and aloe vera extract is potentially useful for cancer and

AIDS. It has been investigated that the aloe vera gel extract has various pharmacological

properties such as promoting and healing wounds and burns, in addition to having anti-

inflammatory, antifungal properties, hypoglycemic and gastro-protective properties

(Reynolds and Dweck, 2019).

1.2 Statement of Problem

Inflammation is a major health concern, contributing to various chronic diseases and medical

conditions. Although synthetic anti-inflammatory drugs are available, they often have side

effects, including gastrointestinal issues, liver toxicity, and immune suppression. Therefore,

there is a growing interest in exploring natural alternatives with minimal side effects.

Despite Aloe vera's extensive use in traditional medicine, there is limited scientific data on its

phytochemical composition and anti-inflammatory potential. The need to evaluate its

bioactive compounds and their efficacy in inflammation management remains a significant

research gap. This study seeks to address this gap by determining the phytochemical

constituents of Aloe vera and assessing it’s in vitro anti-inflammatory activities.

1.3 Objectives of the Study

The primary objectives of this study are:

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 1. To determine the qualitative and quantitative phytochemical composition of Aloe

vera.

2. To evaluate the in vitro anti-inflammatory activities of Aloe vera extracts.

3. To assess the potential correlation between phytochemical constituents and anti-

inflammatory properties.

1.4 Significance of the Study

This study is significant for several reasons:

 Scientific Contribution: It provides empirical evidence on the phytochemical profile

and anti-inflammatory potential of Aloe vera, contributing to the growing body of

medicinal plant research.

 Pharmaceutical Relevance: The findings may aid in the development of natural anti-

inflammatory agents with fewer side effects compared to synthetic drugs.

 Traditional Medicine Validation: It supports the traditional use of Aloe vera by

providing scientific backing for its medicinal properties.

 Economic and Industrial Benefits: The study could enhance the commercial value

of Aloe vera in the pharmaceutical, cosmetic, and nutraceutical industries.

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1.5 Scope of the Study

The study will focus on the determination of phytochemical constituents and the evaluation

of in vitro anti-inflammatory activities of Aloe vera extracts.

1.6 Limitations of the Study

While this study aims to provide valuable insights, certain limitations exist:

 The research is limited to in vitro analysis, which may not fully replicate in vivo

conditions.

 Variability in phytochemical composition due to environmental factors may influence

the results.

 The study does not explore the mechanisms of action of identified bioactive

compounds in inflammation pathways.

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 CHAPTER TWO

LITERATURE REVIEW

1.1 Aloe vera (L) Burn. f.

Aloe vera (L) Burn. is one of approximately 420 species of the genus Aloe, which have

been variously classified as belonging to Aspodeleaceae, Liliaceae or Aloaceae families

(Dagne et al., 2005). Commonly known as Aloe barbadensis Miller, its legitimate name

according to the international rules of botanical nomenclature, is Aloe vera (L) Burn. f.

(Grindlay & Reynolds, 1986). The geographical origin of Aloe vera is believed to be

Sudan, with the plant subsequently introduced to the Mediterranean region and most other

warm areas of the world. The most authentic record of Aloe as a plant with healing

properties is accredited to a Mesopotamian clay tablet dated 2100 BC. However, the first

detailed depiction of the plant’s medicinal value is found in the Papyrus Ebers, an

Egyptian document dated 1550 BC, which sets out multiple Aloe-containing preparations

for the treatment of external and internal ailments. The Aloe vera plant was described in

the Greek herbal of Dioscorides (ca 70 AD), and its use promoted for the treatment of

wounds, hair loss, genital ulcers and haemorrhoids (Davis, 1997). Aloe vera was officially

listed as purgative and skin protectant by the US pharmacopoeia in 1820 (Park & Lee,

2006). It was clinically used in the 1930 for the treatment of radiotherapy burns to the

skin and mucous membrane (Collins& Collins, 1935; Manderville, 1939). Until today,

Aloe is an important herbal medicine in many countries, including China, India, the West

Indies, South Africa and Japan (Grindlay & Reynolds, 1986).

1.2 Habit

Aloe vera is a succulent perennial herb up to 160 cm tall, with a short stem up to 30 cm

long, freely suckering and forming dense groups (Fig 1). Leaves 16-20 in a dense rosette,

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erect to slightly spreading; stipules absent; petiole absent; blade linear - lancelate, 40- 50

cm x 6-7 cm, apex acuminate , margins slightly pinkish with deltoid, firm teeth 2 mm

long, pale, 1-2 cm apart, fleshy, upper surface rather flat, grey green to pale green, lower

surface convex; leaf sap yellowish. Inflorescence terminal dense raceme 30-40 cm x 5-6;

penduncle simple or sometimes 1-2 branched above the middle, 60-90 cm tall; bracts

ovate-acute, deflexed, up to 1 cm long. Flowers bisexual, regular, 3 merous; pedicel c 5

cm; perianth tubular, u to 3 cm long, inflated around the ovary, lobes 6, 3 outer lobes

acute, 3 inner lobes obtuse, yellow, orange or red; stamens 6, exserted; ovary superior, 3-

celled, 6- grooved, style filiform, stigma head- shaped, exserted. Fruit a capsule,

dehiscing loculicadally, many seeded. Seeds are7 mm long, dark brown, winged

(Schmelzer & Gurib-Fakim, 2008)

1.3 Natural habitat

Aloe vera grows in a wide range of climatic conditions. It prefers sandy loamy, well-

drained soil and can grow naturally in poor soils but thrives better in rich soils. It is

tolerant of salinity. Established plants will survive drought quite well even though the root

system is relatively shallow. The plant survives at temperature of -3 0C with only slight

injury. It grows better in full sun or light shade. It is planted as hedge in house premises

and also run wild in the desert conditions and poorest soils Parthipan et al., (2011). Aloe

vera has a habitat variance and is not only in terrestrial habitat but also in rocky habitats

as chasmophytes in the Southern Western Ghats of Coimbatore district of Tamilnadu.

Parthipan et al., (2011) further observed the species as an epiphyte in the Blue Mountains

(2,240 meters above sea level) of

Nilgiri District of Southern Western Ghats of Tamil Nadu.

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Figure 1: Aloe vera

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1.4 Geographic distribution

Aloe vera is only known as a cultivated or naturalized plant. It is generally presumed that

the origin is Arabia, Somalia or Sudan and a recently discovered stand of Aloe vera in

Oman could well prove to be the only population in the world (Afolayan & Adebola,

2006). A Mediterranean origin is often quoted, at present Aloe vera is widely distributed

throughout tropics and subtropics. It is widely grown as a cash crop in dry regions in the

Americas, Asia and Australia. The plant was already used as a drug by the Greeks as early

as 400 B.C. and later by Arabian physician. It is probably present as a cultivated plant in

all countries of tropical Africa (Schmelzer & Gurib- Fakim 2008). Therefore, Aloe vera

present in South Africa is cultivated.

1.5 Folk uses

Aloe vera has been widely used in the treatment of wounds since 550 BC (Shelton, 1991).

For example the Greek physician Dioscorides reported that Aloe vera gel could be used to

heal skin infection, chapping and haemorrhoids (Coats & Ahola, 1979; Gunther, 1934).

There are many reports about the powers of the plant, including that it was the secrete of

Cleopatra’s beauty (Grindlay& Reynolds, 1986) which may account for its use in the

variety of health and beauty aids and that it was applied to the body of Jesus Christ after

his crucification. The species has been widely used in India as a cathartic, stomachic and

anthelminthic, in China as a common folk remedy in most of America and the West Indies

(Boon & Smith, 2004).

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1.6 Main Actions

The active ingredients, whether alone or in concepts, include glycoproteins,

anthroquinones, polysaccharides and low molecular-weight compounds such as beta-sito

sterol (Choi & Chung, 2003).

1.6.1 Wound healing

Wound healing is associated with various mechanisms and constituents of the healing

agent. The omboxane inhibits wound healing and Aloe has been shown to inhibit

thromboxane into vitro (Zachary et al., 1987).Davis, (1989)conducted a study to prove the

whota (Wound Healing, Oral & Topical Activity) of Aloe vera. In his findings, he noted a

62.5% reduction in wound diameter in mice receiving 100 mg/kg/day oral A. vera and a

50.8% reduction was recorded in animals receiving topical 25% A. vera. These data

suggest that A. vera is effective in the treatment of wounds.

Enzymes in Aloe have also been shown to breakdown damaged tissue which can then be

removed by phagocytosis (Bunyapraphatsar et al., 1996). A glycoprotein fraction was

found to increase proliferation of human keratinocytes and increase the expression of

receptors for epidermal grown factor and fibronectin in vitro (Choi et al., 2001). The same

research team then demonstrated that this glycoprotein enhanced wound healing by

increasing cell proliferation invivo. Beta- sitosterol appears to improve wound healing by

stimulating angiogenesis and neovascularisation in vivo (Moon et al., 1999). Aloe

polysaccharides have been shown to eliminate UV-induced immunosuppression

(Strickland et al., 1994).

Several animal studies support the application of Aloe gel to skin damaged by frostbites

as means to maintain circulation and reduce the vasco constrictive effects of thromboxane

10
in the affected dermis (Haggers et al., 1987; Klein & Penneys, 1988; McCauley et al.,

1990; Miller & Koltai, 1995). In combination with the pentoxifylline, it acts

synergistically to further increase tissue survival (Miller & Koltai,1995). Other studies

have found that Aloe gel not only increased collagen content, but also changes collagen

crosslinking, which in-turn increases the breaking strength of scar tissue; making the seal

of wound stronger (Chithra et al 1998; Heggers et al 1996). Aloe vera was found to

promote healing and decreased inflammation in second- degree burns in vivo

(Samboonwong et al., 2000).

Aloe vera prevented delayed hypersensitivity of UV-irritated skin as well as contact

hypersensitivity in animal models with allergic reactions (Strickland et al., 1994).

Acemannan gel (beta- (1, 4 acetylated manna) has demonstrably improved radiation burns

in mice (Roberts &Travis, 1995).

1.7 Use with pharmacological agents

Several topical pharmaceutical antimicrobial agents such as SSD(silver

sulfadiazine)inhibit wound construction, thereby slowing the rate of wound healing. An

experimental model was used to investigate whether co- administration of Aloe could

reverse this effect and improve wound healing rate (Miller et al., 2003). Aloe vera was

found to reverse the delayed wound healing produced by SSD, resulted in the shortest

wound half- life and healing time.

Aloe vera (100 and 300 mg/ kg daily for 4 days) blocked the ability of hydrocortisone

acetone to suppress wound healing by up to 100% (Davis et al., 1997 a). Growth factors

in Aloe vera were thought to mask sterols and certain amino acids that prevent wound

healing. An earlier study identified the sugar mannose -6- phosphate to be one of the chief

constituents responsible for wound healing (Davis et al.,1994 b)

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1.7.1 Ani-oxidants

Studies have shown that several compounds in Aloe gel protect tissues against oxidative

damage caused by free radicals (‘t Hart et al., 1990; Singh et al., 2000; Wu et al., 2006;

Yagi et al., 2002 and Zhang et al., 2006). This is achieved by direct anti-oxidant activity

and indirect activity through stimulation of endogenous anti-oxidant systems. Two aloe

dihydroisocoumarins have been identified and have demonstrated free scavenging

properties (Zhang et al., 2006; Zhang et al., 2008).

Treatment with aloe gel extract decreased lipid peroxidation and hydroperoxides in

diabetic rats to near normal levels (Rajasekaran et al., 2005). The extract also

significantly increased superoxide dismutase, catalase, glutathione peroxidase and

glutathione -S- transferees in the liver and kidney. In another study, data obtained 3.7 and

10 days after exposure to radiation showed that aloe gel significantly reduced oxidative

damage in the liver, lungs, and kidney tissues of irradiated rats (Saada et al., 2003).

1.7.2 Immuno- stimulant

It has been suggested that Aloe may have immune- stimulating capabilities. Much of the

available research has been performed on mice or in vitro and Aloe shows antiviral,

antitumor and non- specific immune-stimulant activity. An experiment in 1980

demonstrated that mice given Aloe extract 2 day before exposure to pathogens were

protected against a variety of fungi and bacteria (Brass et al.,1981). Later the isolated

compound acemannam (beta (1,4)- acelatylatedmannan) was shown to increase the

response of lymphocytes to antigens in vitro (Wamble & Helderman, 1988). In mice,

12
acemannan stimulated cytokines, bringing about an immune- attack on implanted

sarcoma cells, leading to necrosis and regression of cancer cells (Penget al., 1991). A later

trial investigated the effects of acemannan on mouse macrophages

(Zhang & Tizard, 1996). Acemannan stimulated macrophage cytokine production (lL-6

and TNF- alpha, no release, surface molecule expression, and cellular morphologic

changes.

Similarly, a polysaccharide fraction isolated from Aloe vera promoted human

keratinocytes secrete TGF- alpha, TGF- beta- 1, IL- 1- beta, IL- 6, lL-8 and TNF, and

inhibited the release of NO as compared to control (Chen et al., 2005). The immune

enhancing effects of acemannan may be due in part to the compound’s ability to promote

differentiation of

immature dendritic cells (Lee et al., 2001). The cells are crucial for the initiation of

primary immune response.

Multiple studies have highlighted the diverse immunomodulatory activities of Aloe

polysaccharides. Recent research has identified that immunomodulatory activity varies

with size of the polysaccharides entity. It appears that aloe polysaccharides that are of

smaller molecular weight (specifically between 5 and 400 kDa) have the greatest

immunological effects possibly because they are better absorbed than MW entities (Im et

al., 2005).

The purified polysaccharides fraction (PAC- I, PAC- II and PAC-III) from A. vera

stimulated peritoneal macrophages and splenic T and B cells, and increased the ability of

these cells to secrete TNF- alpha, IL- I- beta, IFN- gamma, IL-2 and IL6 (Lueng et al.,

2004). The compound with the highest mannose content and therefore the highest

molecular weight

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(PAC-I), demonstrated the most potential. This finding was again observed by (Lui et al.,

2006) whereby PAC-I exhibited potent stimulation of murine macrophages and produced

tumouricidal properties and activated macrophages in vitro, thus providing evidence in

support of its antitumor properties.

A 99% pure carbohydrate compound (purified acemannan) isolated from Aloe

demonstrated potent haematopoietic and haematopoietic activity in myelosuppresed mice

(Talmadge et al., 2004). Specific manufacturing methods can be applied to enhance the

extracts. For example, 1g of extract obtained from leaves subjected to cold and dark

treatment contained 400mg of neutral polysaccharide compared with 300mg in leaves not

specially treated.

1.7.3 Anti-inflammatory

A number of in vitro and in vivo studies confirm the anti- inflammatory activity of Aloe

vera. The gel reduces oxidation of arachidonic acid, thereby reducing PG synthesis and

inflammation (Davis et al., 1987). It inhibits the production of PGE2by 30% and IL-8 by

20%, but has no effect on thromboxane B2 production in vitro (Langmead et al., 2004).

Following burn injury in vivo, A. vera was found to inhibit inflammation by reducing

leukocytes adhesion and decreasing the pro- inflammatory cytokines TNF- alpha and IL-

6 Duansak et al (2003).

One study conducted on rats with croton oil- induced oedema reported a 47% reduction in

swelling after the application of topical aloe gel (Davis et al., 1987). Another study found

aloe gel to reduce vascularity and swelling by 50 % in the inflamed synovial fluid within

the pouch. When aloe gel was applied topically there was also an increase in fibroblast

cell numbers (Davis et al., 1992). C- glucosylchromone, isolated from aloe gel extracts, is

chiefly responsible for the anti-inflammatory effect, with activity comparable to

14
hydrocortisone in experimental models (Hutter et al., 1996). A study of strptozotocin-

induced diabetic mice further confirmed the anti- inflammatory activity of Aloe vera and

identified the isolated constituent gibberellin as also effective (Davis & Maro, 1989).

Both compounds inhibited inflammation in a dose-dependent manner. A recent in vitro

assessment using human immune cells displaced aloe’s potential to reduce bacteria-

induced pro- inflammatory cytokine production specifically TNF- alpha and IL-1-beta, by

peripheral blood leukocytes stimulated with Shigellaflexneri or lipopolysaccharide (LPS)

(Habeeb et al., 2007a).

1.7.4 Laxative

The Aloe latex contains anthraquinones, which have a stimulated laxative activity. Studies

in rats have shown that aloe latex increases intestinal water content, stimulates mucus

secretion, and induces intestinal peristalsis (Ishii et al., 1994). However, aloe as a laxative

is more irritating than other herbs (Reynolds & Dweck, 1999) and long-term use can

cause an electrolyte imbalance through depletion of potassium salts. Alternatives are

recommended if long- term treatment is required.

1.7.5 Anti-Ulcer

The anti- ulcer of A. vera has been proposed to be due to anti- inflammatory,

cytoprotective, healing and mucus stimulatory effects. According to an in vivo study by

Eamlmnam et al. (2006), [Link] promotes gastric ulcer healing In contrast to these

results, in another study a stabilised fresh aloe gel preparation prolonged the effect of

histamine- stimulated acid secretion but inhibited (Suvtayavat et al., 2004).

15
A number of case reports tell of a positive effect on lung ulcers with to leg ulcers with

topical use of aloe gel, including cases that did not respond to standard medical

interventions (Zawahry et al., 1973). Application of water-based aloe-gel saline soaks,

broad- spectrum antibiotics and antifungals allowed a wound, caused by necrotising

fasciitis, to heal in 45 days in a 72- years- old woman. Aloe gel and saline- soaked

sponges were also used to treat two large seroma cavities caused by deep vein thrombosis

in a 48-year-old man (Ardire, 1997).

Although aloe gel is commonly used as a topical agent for wound healing it is also used

internally. A small study of six patients with chronic leg ulcers found that ingesting 60 mL

aloe juice daily and applying aloe gel directly to the ulcer and surrounding area resulted

in less exudate, odour and seepage through the bandaging (Atherton, 1998).

1.7.6 Hypoglycaemic

Gluconnan slows carbohydrate absorption and slows the postprandial insulin response by

up to 50% (McCarty, 2002). A. vera leaf gel has been investigated as a possible

hepatoprotective and kidney protective agent in diabetes type 2 using animal models. In

one study, the leaf gel and glibenclamide both decreased degenerative kidney changes,

serum urea levels and creatinene levels, but only aloe further reduced kidney lipid

peroxidation (Bolkent et al., 2004). Can et al.(2004) tested aloe pulp, aloe gel extract and

glibenclamide, finding that all treatments decreased liver tissue damage compared to

control animals. Aloe gel extract also increased glutathione levels and decreased non-

enzymatic glycosylation, lipid peroxidation, serum alkaline phosphatase and alanine

transaminase.

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1.7.7 Antimicrobial

A. vera is active against a wide variety of bacteria in vitro, such as Pseudomonas

Klebsiella pneumonia, Strptococcuspyogenes, Staphylococcus aureus and Escherichia coli

(Heck et al., 1981). More recent research indicates antimicrobial activity against

Shigellaflexneri, MRSA, Enterobacter cloacae and Enterococcus bovis (Habeeb et al.,

2007a).

1.7.8 Antiviral

In vitro studies suggest that Aloe vera has antiviral activity due to its interference with

DNA synthesis (Saoo et al., 1996). The polysaccharide fractions of aloe gel inhibit the

binding of benzopyrene to primary rat hepatocytes and thus prevent the formation of

potentially cancer- initiating benzopyrene- DNA adducts in vitro. This was later

confirmed by in vivo studies conducted by Kim &Lee (1997). Moreover, in vitro

experiments have shown the anthraquinones in aloe to be virucidal against HSV 1 and 2,

vaccine viru, para-influenza virus and vasicular stomatitis virus (Anderson, 2003).

Investigation with the acemannan component has identified antiviral activity, particularly

against feline AIDS, HIV type, influenza virus, measles virus and herpes simplex (Kahlon

et al., 1991a and b; Sdydiskis et al., 1991).

1.8 Clinical Use

Although A. vera products are used for many indications, the chief use I treating skin

conditions.

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1.8.1 Skin condition

Aloe is used in the treatment of wound, burns, radiation burns, ulcers, frostbites, psoriasis

and genital herpes, and is the only traditional wound- healing herbal medicine which has

been subjected to a variety of cell culture- based, animal and human- based studies

(Krishnan, 2006). The healing properties may be attributed to antimicrobial, immune-

stimulating, anti- inflammatory and antithrombaxane activities. Allontoin has also been

shown to stimulate macrophage production of IL- 1 and TNF, which are associated with

wound healing (Liptak, 1997).

Most human studies have found that topical application of aloe vera gel increases wound

healing rate and effectively reduces microbial counts; however, there are some negative

studies, most likely related to the fact that the composition of aloe vera gel varies, even

within the same species. Chemical composition depends on source, climate, region, and

the processing method used (Choi &Chung, 2003).

Dry coated aloe vera gloves were tested by 30 women suffering from dry, cracked hands,

with or without contact dermatitis due to occupational exposure; in a contralateral

comparison study carried out byWest & Zhu (2003). Results indicated that aloe vera

glove significantly reduced dry skin, irritation, wrinkling, dermatitis, redness and

improved skin integrity. It would be interesting to see this study repeated using a standard

non-aloe fortified glove on the opposing hand.

The effects of aloe gel applied to skin following dermabrassion in humans are more

controversial, with some patients responding well Fulton (1990), while others have had

severe adverse reactions, including burning sensation and dermatitis (Hunter & Frumkin,

1991). A standard polyethylene oxide gel dressing saturated with stabilised aloe gel was

compared to the standard oxide dressing alone in the study by Fulton and appears to

18
exhibit the strongest evidence overall to support the beneficial effects of aloe vera. The

addition of aloe vera produced a significant vasoconstriction and anti- inflammatory

affect 24 and 48 hours after application. Overall, wound healing was quicker with A. vera

and completed by an average of 72 hours before the oxide gel- treatment. In contrast, one

study found that topical aloe vera gel actually slowed healing after caesaream delivery

(Schmidt & Greenspoon, 1991).

1.8.2 Burns

A recent systematic review of the efficacy of a. vera for healing of burns considered four

controlled clinical trials involving 371 patient. Meta-analysis concluded that topical

treatment with Aloe vera decreased healing time and was specifically more effective for

the first- and second degree rather than the third degree. It was noted that due to

variations in the Aloe preparations and outcome measures used in the studies, more

specific conclusions and cannot be drawn (Maenthalsong et al., 2007).

One study involving 18 outpatients with moderate to deep second degree burns aging

from 2 to 12% of total body surface area showed that commercial aloe vera ointment was

as affective as SSD in regard to protection against bacterial colonisation and healing time.

More specifically, the mean healing time with aloe vera treatment was 13 days compared

with 16.15 days for SSD (Heck et al.,1981).

Results are less encouraging for sunburn protection and healing. A randomised double-

blind trial in 20 healthy volunteers evaluated the effect of aloe vera cream for both

prevention and treatment of sunburn (Puvabanditin & Vongtongsri, 2005). The cream

(70% aloe) was applied30 minutes before, immediately after, or both before and after UV

irradiation. The cream then continually applied daily for 3 weeks. The results showed that

the aloe vera cream did not protect against sunburn and was not an effective treatment.

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1.8.3 Frost Bite

In combination with other treatments, topical A. vera significantly enhances healing and

has a beneficial effects of topical aloe vera cream in combination with standard treatment,

such a rapidly re-warming the affected areas, analgesics, antibiotics and debridement (n=

56) with another group of 98 patients who did not receive A. vera treatment. Of those

receiving A. vera in addition to usual treatment, 67% healed without tissue loss compared

with 32.7% in the group control. Additionally, 7.1% of the total group of 56 required

amputation compared with 32.7% in the control group. Although encouraging, this study

is difficult to interpret because the groups were not well matched and combination

therapies differed (Heggers et al., 1987).

1.8.4 Radiation- induced dermatitis

A recent reviewed concluded that aloe gel was as mild steroids creams, such as 1%

hydrocortisone, in reducing the severity of radiation burn, without the side-effects

associated with steroid creams (Muddocks-Jennings et al., 2005). In contrast, another

review concluded that aloe was ineffective for the prevention or reduction of side-effects

to radiation therapy in cancer patients (Richardson et al., 2005). That review analysed 1

past review, 5 published RCTs. It is important to note that various preparations such as

cream, juices, gels and fresh aloe had been tested, which makes it difficult to assess the

evidence.

1.8.5 Psoriasis

A double-blind placebo-controlled study found topical aloe vera extracts 0.5% in a

hydrophilic cream can be beneficial in the treatment of psoriasis and PASI (psoriasis and

severity index) scores between 4.8 and 16.7 (mean 9.3) participated in the study, which

20
was scheduled for 16 weeks with 12 months of follow-up. Patients were examined weekly

and those showing a progressive reductive of lesions, desquamation followed by

decreased erythema, infiltration and lowered PASI score were considered healed. By the

end of the study, Aloe vera extract cream had cured 83.3% of patients compared with the

placebo cure rate of 6.6% (P< 0.001). PASI scores decreased to a mean of 2.2 (Syed et al.,

1996a). In contrast, a randomised, double- blind, placbo- controlled trial found no

significant benefits with a commercial aloe vera gel in 41 patients with stable plaque

psoriasis (Paulsen et al.,2005). Following a 2- week washout period patients applied

either the aloe gel or placebo twice daily for 1 month. Redness and desquamation

decreased by 72.5% in the active treatment group as compared to 82.5% in the placebo

group. It should be pointed out that 82.5% is an extremely high placebo responder rate.

Fifty-five per cent of patients reported local side-effects, mainly drying of the skin on test

areas.

1.8.6 Genital Herpes

Two clinical studies have investigated the effects of Aloe vera 0.5% topical preparations

in genital herpes, producing good results. A double- blind, placebo-controlled study has

demonstrated that aloe vera extract 0.5% in a hydrophilic cream is more efficacious than

placebo in the treatment of initial episodes of genital herpes in men (n= 60, aged 18-40

years). Each patient was provided with a 40 g tube, containing placebo or active

preparation of trial medication to their lesions three times daily for 5 consecutive days

(maximum 15 topical application per week). The treatment was well tolerated by all

patients (Syed, 1997). The other study involving 120 subjects used a preparation

containing 0.5% of whole aloe leaf extract in a hydrophilic castor and mineral oil cream

base, which was applied three times daily 5 days per week for 2 weeks. Treatment

resulted in a shorter mean duration of healing compared with placebo, Aloe cream also
21
increased the overall percentage of healed patients and there were no significant adverse

reactions reported (Syed et al., 1996b).

1.8.7 HIV

The acemannan component of Aloe vera has been used as adjunctive therapy to

antiretroviral therapy in HIV infection. A preliminary clinical trial found that acemannan

may enhance the activity of the anti-HIV drug AZT. A dose of 800 mg acemannan daily

significantly increased circulating monocytes (macrophages) in 14 HIV patients. Aloe

increased the number and activity of the monocytes (McDaniel et al.,1990). Subsequently,

a randomised, double-blind placebo-controlled study of 63 male subjects with advanced

HIV, taking zidovudine and didanosine, investigated the effects of 400 mg of acemannan

taken four times daily for 48 weeks. Results showed a decrease in CD4 cell numbers in

the acemannan group compared with placebo (Montaner et al., 1996).

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 CHAPTER THREE

MATERIALS AND METHODS

3.0 SAMPLES COLLECTION

Alove vera (aloe barbadensis miller) will be collected from Federal Polytechnic Mubi garden

Adamawa State, and all the chemical use for the chemical analysis will be obtained from the

Department of Chemical Science and Technology Federal Polytechnic Mubi Adamawa State.

3.1 Extraction of plant material for qualitative analysis

For the qualitative analysis, a fresh sample of aloe vera leaves will be cut into small pieces.

leaves will be crushed with a pestle and mortar. Fifty grams (50g) each will be weighed into

three different Erlenmeyer flasks (250mL each) and labeled as samples A, B, and C. Hundred

milliliter (100 mL) of diethyl ether will be added to sample A, 100 mL of ethanol will be

added to sample B, and a 100 mL of distilled water will be added to sample C, and the three

samples were corked. The leaves were soaked in these solvents for 24 hours, decanted and

heated to concentrate the plant extract. The aqueous plant extract will be filtered using a fine

muslin cloth and the solutions will be extracted. The extract concentration of the resulting

filtrate was 100%. The standard methods for the identification of the components of the

samples were adopted (Maan et al., 2018).

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3.2 Extraction of plant material for quantitative analysis

For the quantitative determination, fresh leaves of the Aloe vera plant will be collected and

dried at room temperature and then crushed into powder using laboratory sterile mortar and

pestle. The dried powdered extract will be then further use in the determination of the

quantity of the phytochemicals present in the plant. This method will be reported in a study

conducted by Usman et al., (2020).

3.3 The Qualitative Phytochemical Screening of Aloe vera Leaf Extracts

Determination of alkaloids

About 2 mL of each extract sample will be stirred with 2 mL of 10% hydrochloric acid. A 1

mL portion will be treated with a few drops of Wagner's reagent and a second 1 mL portion of

the extract will be similarly treated with Meyer's reagent. Presence of alkaloid was confirmed

by HPLC method (Mukherjee et al., 2013).

Determination of flavonoids

Sodium hydroxide (NaOH) solution (10%) will be added to 3 mL of filtrate of each extract

sample. The yellow color observed indicates the presence of flavonoid compounds (Bibi et

al., 2021).

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Determination of tannins

A solution of 0.1% ferric chloride (FeCl3) was added in drops into 5 mL of extract after it has

been boiled with 20 mL of CHCl 3, the appearance of brownish colour shows the presence of

tannin (Bibi et al., 2021).

Determination of saponins

About 5 mL of the aqueous extract will be taken and heated with 5 mL of distilled water and

will be filtered. Few drops of olive oil will be added to 10 mL of the filtrate. Emulsion

formation will be taken as an indication of the presence of saponins (Bibi et al., 2021).

Determination of glycosides

A two milliliter (2 mL) solution of 50% H 2SO4 will be added to 5 mL of the extract in a test

tube. The mixture will be heated in boiling water for 15 minutes. It will be cooled,

neutralized with 10% NaOH, 5 mL of Fehling’s solution will be added and the mixture will

be brought to a boil. A brick-red precipitate will be observed, indicating the presence of

glycosides (Bibi et al., 2021).

Determination of anthraquinones

Ten milliliters of benzene and 5 mL of 10% ammonia solution will be added to 0.5g of the

extract. The mixture will be shaken and the presence of pink, red or violent color in the

bottom indicates the presence of anthraquinones (Bibi et al., 2021).

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3.4 The Quantitative Phytochemical Screening of Aloe vera Leaf Extracts

Alkaloid Determination

Alkaloid content will be determined by following the method used by Luyang et al., (2015).

Five grams of powdered plant extract will be added to 100 mL of methanol water (1:1). The

mixture and solvent will be evaporated. The resultant residue will be mixed with 20 mL of

0.0025M H2SO4 and partitioned with ether to remove unwanted materials. A strong base

(NH3) will be added to the aqueous fraction solution, followed by extraction with excess

chloroform to obtain the alkaloid fraction or separated by filtration. The chloroform

extractions will be repeated several times and the extracts will be concentrated to dryness.

The alkaloids will be weighed and the percentages will be calculated relative to the initial

weight of the powder.

% Alkaloid= weight of the alkaloid residue × 100

Weight of sample

Determination of tannins

Tannins content will be estimated by following the method used by Muhammad et al.,

(2012). A powdered sample (0.1) of the plant extract was placed in a 100cm 3 Erlenmeyer

flask and a 50cm3 volumetric flask. The residue will be washed several times and a mixed

solution of distilled water containing 0, 1, 2, 3, 4 and 5cm 3 of standard tannic acid and 10cm3

26
of Na2CO3 solution will be added and made up to volume with distilled water. The optical

density will be

% Tannin = absorbance of samples × average gradient factor × dilution factor ×100

Weight of samples ×100

Determination of saponins

Saponins content will be estimated by following the method reported by Chinelo et al.,

(2013). Five grams of powdered plant extract will be added to a 250 mL flask containing 30

mL of 50% alcohol. The mixture will be refluxed for 30 minutes and immediately filtered hot

through coarse filter paper. Two grams (2g) of charcoal will be added. The contents will be

boiled hot and filtered. The extract will be cooled (some saponins could be isolated) and an

equal volume of acetone will be added to complete the saponin precipitation. The isolated

saponins will be collected by decantation, dissolved in a minimal amount of boiling 95%

alcohol and hot filtered to remove insoluble material. The saponin precipitated when the

filtrate will be cooled to room temperature. The separated saponins will be collected by

decantation, suspended in about 2 mL of alcohol and filtered. The filter paper will be

immediately transferred to a desiccator containing anhydrous calcium chloride to dry the

saponin. They will be weighed for the extract used.

% saponin = Weight of dried extract × 100%

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 Weight of sample

Determination of glycosides

Glycosides content will be determined by following the method adopted by Shamsu and

Abubakar (2016). One gram (1g) of the extract will be added into 10 mL of 70% alcohol and

the mixture will be filtered. From the filtrate, 8 mL of the mixture will be added to 8 mL of

12.5% lead acetate to precipitate resins, tannins and pigments. The mixture will be shaken

well, made up to another volume of 100 mL with distilled water and then filtered. The filtrate

(50 mL) will be pipetted into another 100 mL volumetric flask and 8 mL of 4.7% disodium

hydrogen phosphate (Na2HPO4) solution (to precipitate excess lead) will be added. The

mixture will be made up to volume with distilled water and mixed. The mixture will be

filtered through Whatman filter paper. 10 mL of Baljet's reagent will be added to 10 mL of

the purified filtrate. A 10 mL blank of distilled water will be added to 10 mL of Baljet

Reagent. The two will be left undisturbed for 1 hour (maximum coloring time). Color

intensity will be read at 495nm against a blank (20 mL distilled water) using a

spectrophotometer. The color remained stable for several hours. The percentage of total

glycosides in digitoxins will be simply calculated using E¹cm 1% of given digitoxins (which

is equivalent to 170).

% Glycoside = A
17 ×100×g%
Where A= Absorption of the color at 495nm

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