Republic of the Philippines

Department of Education
Region V
Tabaco National High School
Tabaco City

Phytochemical Analysis and Pharmaceutical

Evaluation of Malubago

Micah Atondo
Angelica Joyce Calibo
Ian Arvin Bellen
John Marc Carlet
Researchers

Mr. Ronaldo Reyes

Research Adviser
 ABSTRACT

Wound healing is part of the body's natural process of self-repair and of regenerating skin

tissue. However, advance medical technologies enable faster wound healing. But there are

increasing cases in which microbial strains adapted and gain antibiotic resistance. We formulated

a study through which we can have alternatives for these herbal creams. This study will fill the

gap of lacking alternatives to multi-resistant microbial strains. It aims to identify the

phytochemical constituents of Malubago leaves and to evaluate and select the best formulation

based on different pharmaceutical parameters. We conducted series of tests to determine the

potential of Malubago leaves as an alternative for herbal creams. To determine its chemical

constituents, we submitted leaves to the Development Institute- Standards and Testing Division of

the Department of Science & Technology (DOST- ITDI). Knowing its chemical constituents, we

prepared formulations of herbal cream from Malubago leaves with same contents but with different

concentrations. Pharmaceutical evaluation of herbal cream was conducted through testing the pH,

homogeneity, stability and spreadability. The product has met the requirements needed in the

pharmaceutical evaluation. Based on the phytochemical analysis of malubago leaves and

formulations that we prepared, we can conclude that malubago leaves have a potential to be an

alternative for herbal cream.

 Chapter I

INTRODUCTION

Background of the Study

A wound can be defined as a disruption in the continuity of the skin or mucosa. Wounds,

which occur most commonly due to accidents such as burns, can be broadly classified as acute or

chronic wounds. Acute wounds normally heal in a short period of time provided the right treatment

is given; on the other hand, chronic wounds take a longer period of time to heal or sometimes even

recur due to underlying pathology. In wound healing, it is always a must to achieve rapid and

complete resolution; hence, the goal of every medical practitioner is to achieve early wound

healing.

Due to the rise in antibiotic resistance, traditional or herbal medicines are increasingly

becoming an alternative to overcome multi-resistant microbial strains. These also help to

significantly reduce financial burden.

Herbal Medicine, also called botanical medicine or phytomedicine, refers to using a plant’s

seeds, roots, leaves, bark, or flowers for medicinal purposes. It is becoming more prevalent as

improvements in quality control and accurate results, along with advances in chemical research,

showcased its benefit in treating and preventing diseases. Recently, the World Health Organization

(WHO) estimated that 80% of people worldwide depend on herbal medicine as one of their primary

health care.

Hibiscus tiliaceus Linn., which belongs to the Malvaceae Family is an evergreen coastal

tree that grows 4 to 12 meters high. In many places, the tree can be seen growing as a coastal wind-

break, a hedge or a boundary plant, and aliving trellis or fence post. Its wood is also use for building
canoe parts, crafts, and as a fuel wood, but above all, it is widely utilized as a medicinal plant

(Elevitch and Thomson, 2006). As matter of fact, many scientific studies have already been

conducted on its medicinal properties, such as its anti-inflammatory (Borhade et al., 2012), anti-

microbial (Hemaiswarya et al., 2009), anti-tumor (AnbuJebaSunilso et al., 2008), antioxidant

(Ramprosahd et al., 2012), anti-ulcer (Sharma, 2010), anti-tyroxinase (Wong et al., 2010), anti-

depressant-like (Vanzella et al., 2012), anti-genotoxic and anti-mutagenic (Rosa et al., 2007)

effects. However, none ventured into its capacity to heal wounds despite the fact that its ethanol

extract has successfully showed an activity against the gram-positive bacterium Staphylococcus

aureus, and the gram-negative bacteria, namely Escherichia coli and Salmonella paratyphi.

Statement of the Problem

The researchers would like to evaluate the potential of herbal cream out of Malubago

(Hibiscus tilliaeus Linn.) leaves in healing wounds.

Objectives

Specifically, the study seeks to:

1. Identify the phytochemical constituents of Malubago leaves;

2. Evaluate and select the best formulation based on different pharmaceutical parameters, which

will include appearance, pH, homogeneity, viscosity, stability and spreadability;

Hypothesis

1. No best formulation of herbal cream can be derived from the study.

2. Herbal cream from Malubago leaves is not safe to use.

Scope and Delimitation

The whole study will be conducted from August to December, 2016 at the laboratory and

animal house facilities of Tabaco National High School (TNHS) Science Laboratory, Tabaco City.

The study will focus on screening Malubago leaves as a potential wound healing cream; therefore,

it will not include other parts as well as its other benefits given by the plant except for its young

leaves and likelihood to help achieve complete wound healing.

The study didn’t focus on the effect of Hibiscus Tiliaceus Herbal Cream (HTHC) to actual

test subjects. Likewise, the effect of shelf-life of the herbal cream is beyond the scope of the study.

Significance of the Study

This study was conceptualized to characterize and identify H. tiliaceus L. as a contribution

to the rich store of potential alternative medicines that will be able to effectively achieve rapid,

complete and early wound healing. Specifically, the study aims to benefit the following:

Department of Health (DOH). This study will entail the attention of the Department of

Health, particularly in terms of use of medicinal alternatives or natural extracts.

Research Institute for Tropical Medicine (RITM). The success of the study may provide

a preliminary data in giving information, education, and advocacy on the potential application of

H. tiliaceus L. on wound healing.

Malubago Farmers. This will provide local jobs for malubago farming.

Students and Researchers. This will be a database for future wound healing researches

and ointment development.

 Chapter II

REVIEW OF RELATED LITERATURE AND STUDIES

Related Literature

The skin is the largest organ in the human body, comprising approximately 15% of total

adult body weight. The skin maintains our internal environment while protecting us from the

external environment and it allows us to experience a wide range of stimuli, from pleasure to pain.

Any break in this continuity of the skin surface is the first step in the formation of a wound and

provides a potential portal of entry for infection.

Specifically, a wound is a break in the progression of body structures caused by violence,

trauma, or surgery to tissues. It is an injury to the body that typically involves laceration or

breaking of a membrane and usually damage to underlying tissues. A wound can be as simple as

a surface abrasion, or it can be an extensive, life-threatening destruction of tissue that reaches down

to and includes the internal organs of the body.

Generally, wounds can be acute or chronic. Acute wounds are traumatic or surgical wounds

that usually heal over time according to the normal wound-healing process. They often vary, and

can be just scratches or deep wounds with inconsistent amounts of tissue loss and damage to blood

vessels, nerves, muscles, other tissues, or internal organs. On the other hand, wounds are defined

as chronic when they have failed to proceed through an orderly and timely healing process, thereby

unable to establish a sustained anatomic and functional result. Usually, healthy people do not

manifest chronic wounds; instead, they are very often related to a condition like diabetes mellitus.

There are many different types of wound, but only a few important ones will be mentioned

here. A gunshot wound is a penetrating injury from a bullet shot from a gun. At very close range,
the wound may have gunpowder deposits and the skin burn marks. The severity of the wound may

depend on the structures damaged, the velocity and caliber of the bullet, and the underlying health

of the victim. A puncture wound is a wound made by a sharp-pointed instrument such as a dagger,

ice pick, or needle. A puncture wound usually is collapsed, which provides ideal conditions for

infection. The placement of a drain, anti-tetanus therapy or prophylaxis, and gas gangrene

prophylaxis may be required and severity usually depends on the nature of the instrument that

caused the injury. An abdominal wound is a traumatic injury or surgical incision which may be

superficial or can extend to intraperitoneal or extraperitoneal organs or tissues. In cases of

abdominal trauma, a careful examination (often including peritoneal lavage, ultrasonography, or

computed tomographic scanning of the abdomen) is necessary to determine the precise nature of

the injury and the proper course of treatment. Superficial injuries may require no more than

ordinary local care, however, when major bleeding or organ damage has occurred immediate

laparotomy may be needed. Intravenous fluids, blood components, antibiotics, and tetanus

prophylaxis can also be given when necessary.

Restoration of integrity to injured tissues, such as wounds, by replacement of dead tissue

with viable tissue is coined as wound healing. Wound healing usually starts immediately after an

injury, but may continue for months or years, and is essentially the same for all types of wounds.

Variations are the result of differences in location, severity of the wound, extent of injury to the

tissues, the age, nutritional status, and general state of health of the patient.

Wounds also heal in one of two possible ways: regeneration or scar formation. In

regeneration, the tissue that has been destroyed or damaged is replaced by tissue of the same type.

This is the preferable way for wounds to heal because it preserves proper functioning of the injured

site and its normal appearance. In scar formation healing, the lost tissue is replaced by fibrous scar
tissue, which does not have the same properties as the original tissue and is unable to carry out the

same functions (Doughty & McNichol, 2015).

However, due to increasing incidence of microorganism’s resistance to antibiotic, wound

healing can now become increasingly challenging, that is why, traditional or herbal medicines are

becoming an alternative to overcome multi-resistant microbial strains and to significantly reduce

financial burden.

Malubago (H. tiliaceus Linn.) is found throughout the Philippines, along the seashore and

tidal streams. It is occasionally planted inland for ornamental purposes, but is likewise used as

herbal plants and is considered aperitive, emollient, emetic, diuretic, febrifuge, sudorific, and

laxative. Its fiber is used to make a strong rope, string and hog traps. It is also known for its ability

to protect river banks (or shorelines) from erosion; it can repair itself quite well, and is extremely

resilient. It is also proven to have anti-inflammatory, anti-tumor, anti-depressant, and anti-

mutagenic properties. (Stuart, 2016)

Related Studies

The Herbal External Healing Cream or Balm is for external injuries where the skin is

broken and/or allergy prone and is in need of soothing and healing. It can be used for cuts, scrapes,

grazes, eczema, nappy rash, perineal tears after birth or caesarean scars. It contains Calendula

which is one of nature’s fastest healers, Comfrey which is healing and has anti-inflammatory

properties, and Chickweed which is very soothing and is great for red, itchy rashes or eczema. The

essential oils Lavendar, Chamomile and Geranium are very soothing and smell great while Carrot

Seed oil contains high levels of Vitamin A to help with healing.

 In the study entitled “Experimental Evaluation of a Herbal Cream (SK-AC) On Acetylated

Lanolin Alcohol-Induced ACNE In Rabbit”, experimental studies were conducted in four groups

of rabbits using external ear canal induction of comedogenesis and its correction by a herbal

formulation, viz., SK-AC cream. The result brought out that the SK-AC cream restricted the

comedogenesis induced by acetylated lanolin alcohol when compared with other defined groups.

Outcome of this experiment pointed out the usefulness of SK-AC cream for the treatment of acne.

(Mitra et al., 1998)

Herbal ointment containing methanol leaf extract of Jatropha curcas L. was formulated

and tested for pro-wound healing activities. The extract (0.5, 1.0. and 1.5 g) was incorporated into

10 g of a simple ointment base by melting and trituration to give three batches of the ointment

formulation. Excision wound measuring about 177 mm2 was created on the albino rats placed in

groups (n = 5) and the ointment applied topically on the wounded area which was measured at

intervals of 3 days until epithelialization and complete wound closure. Blank ointment base and

Gentamicin ointment (1 %) served as the control and standard treatments, respectively. Topical

application of the ointment caused a significantly (P<0.5) higher rate of wound healing and

reduced the epithelialization period in a dose–related manner. Application of the ointment batch

containing the highest concentrations of J. curcas extract (1.5 g/10g ointment) showed the highest

rate of wound closure reducing the epithelialization period to 14.8 days compared to the blank

ointment treatment with epithelialization period of 18.8 days. The study concluded that

formulating J. curcas extract as ointment is effective in wound care and should be explored in

harnessing the potentials of the plant in the treatment of topical diseases.

Lantana camara Linn. is a heavily branched shrub that can grow in compact clumps, dense

thickets or as a climbing vine that has covered large areas in India, Australia, and much of Africa.
In order to evaluate the wound healing activity of Lantana camara L., two different solvent

extracts were prepared from the leaves of the plant. Ethyl acetate and ethanol were used for the

extraction of the active ingredients. Excision wound model on Wistar albino rats of 4 months, of

both sexes, weighing between 100 to 150 g was used to assess the wound healing activity of the

leaves. Results showed that remarkable wound healing activity was observed with the ointment

formulation of the ethanol extract at 2% concentration. Wound contraction was calculated as

percentage of the reduction in wound area. A specimen sample of tissue was isolated from the

healed skin of each group of mice for the histopathological examination and the results of which

also supported the outcome of excision wound models. The study thus demonstrated that the aerial

parts of Lantana camara L. promoted wound healing activity in mice. (Shonu, 2012)

The blood-red sap of Dragon's blood has been used in folk medicine for fractures, wounds,

inflammation, gastrointestinal disorders, rheumatism, blood circulation dysfunctions, and cancer.

Existing in vitro and in vivo bioactivity of this herb on different mechanisms of healing shows

strong potential of this sap in wound healing. In fact, a clinical trial entitled “Efficacy of a Dragon’s

Blood Cream on wound healing: a randomized, double-blind, placebo-controlled clinical trial”,

was designated to evaluate the wound healing effect of Dragon's blood on human wounds. Sixty

patients, between the ages of 14-65 years, who were referred to remove their skin tag, were

assigned to this double-blind, placebo-controlled, randomized clinical trial and received either

Dragon's blood or a placebo cream. They were visited on the 3rd, 5th, 7th, 10th, 14th, and 20th

day of the trial to check the process of healing and to measure the wound's surface. At the end of

trial, there was a significant difference in the mean duration of wound healing between the two

groups (p = 0.0001). The phenolic compounds and the alkaloid taspine, which exist in Dragon's-

blood resin, are probably the main reasons for the wound healing property of this plant. Being
naturally accessible, safe and affordable makes Dragon's blood cream a good choice for addition

to the wound healing armamentarium. Further studies on wounds with different causes and among

larger populations are suggested to ensure the effectiveness and safety of Dragon's blood.

Synthesis of the Art

Malubago (Hibiscus tiliaceus) has been proven of having properties capable to be one of

the plants effective in curing illnesses like colds, fever, and also ulcer (Sharma, 2010). Researchers

had also tested the plant’s significant effect as an anti-Microbial (Ramproshad, et al., 2012).

Relating this to the study, since Malubago is proven to have anti-microbial properties, the

researchers had decided that Malubago can be used for producing an alternative herbal cream for

wound healing.

Gap to be bridged

Many researchers have been using different kinds of plants in making herbal creams or

ointments but none of them used Malubago (Hibiscus tiliaecus Linn.) plant. Regarding the above

mentioned studies that typically revolves around different plants except the Malubago plant, this

study focuses entirely on the leaf and its healing potency. The other studies focuses on the use of

commercialized oils and emulsifying agents, whereas the researches will use all natural ingredients

in making the herbal cream. This is what the study wants to attain that other studies didn’t do.
 Chapter III

METHODOLOGY

Collection and Identification of H. tiliaceus L.

Using polyethylene bags, average-sized young Malubago leaves were collected from San

Lorenzo, Tabaco City. Selection of the leaves was made on the basis of absence of leaf spots at

the time of collection. The leaves were verified by the Officer-in-Charge of the Department of

Agriculture Tabaco City, Ms. Armi J. Brobio as Malubago (Hibiscus tiliaceusLinn.) leaves.

Phytochemical Analysis of the H. tiliaceus L.

Two hundred fifty (250) grams of Malubago leaves was submitted to the Development

Institute- Standards and Testing Division of the Department of Science & Technology (DOST-

ITDI) in Bicutan, Taguig City for the determination of its chemical constituents.

Preparation of Methanolic Extract

The leaves were washed with tap water and were air dried for 5 days. Homogenization of

the leaves into coarse powder was carried out using a mortar and pestle. Homogenized leaves were

then soaked in 95% analytical-grade methanol (1 g: 10 mL) for 5 days and the resulting mixture

was filtered through Whatman® filter paper No. 1. Then, the filtrate obtained from the mixture

was subjected to simple distillation process to separate the excess methanol content of the solution.

Cream Formulations

Stearyl alcohol and white petroleum were first melted together using water bath at about

75˚C. Then, the oil obtained in methanolic extract was dissolved in purified water to obtain the

different concentration of 5, 10, 20 %w/w. Each concentration was added separately with stirring
until the mixture congeals and beeswax was added next to act as an emulsifying agent. Lastly,

methyl paraben was added as a preservative and the mixture was cooled under stirring and a soft

mass of cream is obtained. Table 1 shows the prepared formulations of herbal cream with

methanolic extract of Hibiscus tiliaceus L.

Table 1. Prepared Formulations of Herbal Cream from Malubago Leaves

Formulations Ingredients Concentration (%w/w)

HTHC I Oil Extract 5
Stearyl alcohol 25
White petroleum 25
Beeswax 1
Methyl paraben 1 drop
HTHC II Oil Extract 10
Stearyl alcohol 25
White petroleum 25
Beeswax 1
Methyl paraben 1 drop
HTHC III Oil Extract 20
Stearyl alcohol 25
White petroleum 25
Beeswax 1
Methyl paraben 1 drop
HTHC: Hibiscus tiliaceus Herbal Cream

Pharmaceutical Evaluation of Herbal Cream

All evaluation parameters will be carried out in all formulations at different time intervals,

namely the 15th, 30th, and 60th day.

 pH. 0.5g of each formulation was dispersed in 50 ml of distilled water and the pH was

determined using a digital pH meter (Panigrahi et al., 1997).

Homogeneity. All of the ointment formulations produced was tested for homogeneity

based on its visual appearance. Homogenous ointments were described as those with uniform color

and have no lumps (Panigrahi et al., 1997).

Stability. This was performed by exposing all formulations at different temperature

conditions (4, 25, and 37˚C).

Spreadability. This was determined by using the formula S = (M)(L)/T where S is

spreadability, M is weight tied to upper slide, L is length of glass slide, and T is time taken to

completely separate the slides from each other. The lesser the time taken for the two slides to

separate, the better is the spreadability.

 Chapter IV

RESULTS AND DISCUSSIONS

Phytochemical Study of Malubago Leaves Extract

Basing from the phytochemical analysis conducted by the Industrial Technology

Development Institute – Standards and Testing Division of the Department of Science &

Technology (DOST- ITDI) in Bicutan, Taguig, the abundant chemical constituents present in

malubago leaves are sterols and glycosides (Table 1). There are also moderate amount of saponins

and traces of flavonoids. According to the literature one or more of the major constituents of the

plant like the flavonoids and triterpenoids may possibly account for the observed antimicrobial

activity of the plant extract (Pradeep et al., 2009)

Table 2. Phytochemical Study of Malubago Leaves

Sterols (+++)
Triterpenes (-)
Flavonoids (+)
Saponins (++)
Glycosides (+++)
Tannins (-)
Alkaloids (-)
Legend: (+) Traces, (++) Moderate, (+++) Abundant, (-) Absence of constituents

Sterol is any steroid-based alcohol that contains hydrocarbon (aliphatic) side-chain

of 8-10 carbons at the 17-beta position and a hydroxyl group at the 3-beta position. Sterols in plants

are called phytosterols, and it has the same chemical structure as cholesterol. The differences

between them arise from the lateral chain which is modified by the addition of one or two

supernumerary carbon atoms at C-24. The 24-alkyl group is characteristic of all phytosterols and

is reserved during subsequent steroid metabolism in both fungi and plants to give hormones that
regulate growth and reproduction in a manner similar to animals (Leray, 2014). Plant sterols and

stanols are substances that occur naturally in small amounts in many grains, vegetables, fruits,

legumes, nuts, and seeds. Since they have powerful cholesterol-lowering properties, manufacturers

have started adding them to foods.

Glycosides are a class of molecules in which, a sugar molecule is bonded to a "non-sugar"

molecule. Glycosides play important roles in our lives. Many plants store medicinally important

chemicals in the form of inactive glycosides. The non-sugar portion contains the biochemically

active properties of medical interest. Once the glycoside is split into its two components (sugar

and non-sugar parts), the non-sugar component is now free to exert its chemical effects on the

body. For example, digitalis is a glycoside that when ingested, causes the heart to contract (pump)

more forcefully. This is useful in medicine, where heart failure is present.

Saponins are group of naturally occurring plant glycosides which are characterized by their

strong foam-forming properties in aqueous solutions. Due to the great variability of their

structures, saponins always display anti-tumorgenic effects through anti-tumor pathways.

(Elsevier, 2010)

Flavonoids are group of plant metabolites thought to provide health benefits through cell

signalling pathways and by virtue of its antioxidant effects. Flavonoids are found in a variety of

fruits and vegetables. Some flavonoids also inhibit certain spores to protect against certain plant

diseases. Flavonoids are ubiquitous in plants and are the most common type of polyphenolic

compound found in the human diet. The abundance of flavonoids coupled with their low toxicity

relative to other plant compounds means they can be ingested in large quantities by animals,

including humans. Examples of foods that are rich in flavonoids include onions, parsley,

blueberries, bananas, dark chocolate and red wine (Mandal, 2014).

Pharmaceutical Evaluation of Herbal Cream

HTHC II was found to be the best and satisfactory compared to all other formulations. It

had light green appearance and gave a smooth feel on application. This was maintained even after

subjecting the herbal cream in the stability study. The pH of the formulation was found to be 6.50

which was near the optimum pH for a good skin, which is 6.8. The cream also showed good

spreadability (15.90 g/sec) when measured during slide. After application of the cream, the type

of smear formed was non-greasy and was easily removed on washing with tap water

CONCLUSION AND RECOMMENDATIONS

Conclusions

The researchers had found out the potential of Malubago Herbal Cream (HTHC) as an

alternative healing cream for wounds. The results of the pharmaceutical evaluation of HTHC was

dependent on the concentration of the natural ingredients in making the herbal cream. The most

effective formulation to be used is the HTHC II which has the adequate concentration of each

ingredient used.

With all the promising pharmaceutical results obtained, it is therefore concluded that HTHC

malubago (H. tiliaceusL.) leaves can become a potential source in the manufacture of healing

herbal cream.
Recommendations

1. Further study on the healing properties of the malubago (H. tiliaceusL.) leaves be

investigated to test its effectiveness to actual test subject.

2. Isolation of the actual chemical components of the plant with healing potencies be further

carried out.

3. Other parts of the malubago (H. tiliaceusL.) leaves be further studied to test its

effectiveness as a potential wound healing agent.

4. The effect of shelf-like of the healing herbal cream be further studied.

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DOCUMENTATION