CHAPTER 1

THE PROBLEM AND ITS BACKGROUND

Introduction

Bacteria, fungi, viruses and nematodes are microorganisms that predominantly

cause human infections in tropical and subtropical countries.

Bacterial infection is any type of human infection that is caused by bacteria rather

than a virus. Bacteria are very common in the body and in the surroundings. They are

everywhere and usually in infinite quantities. Some of them are helpful, some are

neutral. Less than 1% of these bacteria are harmful which give rise to bacterial infection.

Bacterial infections are usually treated with antibiotics. The specific type of

antibiotic depends on the type of bacteria that is causing the infection. The type of

medication is based on the symptoms and diagnosis demonstrated by the patient.

Recently, antibiotic resistance has become a global concern as the clinical efficacy of

many existing antibiotics is being threatened by the emergence of multi – drug –

resistant pathogens. 

Over the last centuries, intensive efforts have been made to discover clinically

useful antibacterial drugs. Green plants possess the broadest spectrum of synthetic

activity and have been the source of many useful compounds like flavonoids, alkaloids,

tannins and other phenolic compounds which have been established as the most

important bioactive compounds so called secondary metabolites. Among these

secondary metabolites are tannins which are water-soluble polyphenolic substances

and that usually occur in leaves in great quantities.

There are many local plants that contain tannin. One of them is Bauhinia

malarabica from the Family Fabaceae, commonly known in Tagalog as

“Alambangbang” and “Alibangbang” in Ilongo is widely distributed in Luzon. Bauhinia is

a genus of more than 200 species. The genus was named after the Bauhin brothers,

Swiss-French botanists. The species share the 'butterfly' configuration of the leaves.

Studies shows that Alambangbang has potential benefits as anti-inflammatory,

antinociceptive, antipyretic and antioxidant activity. (Stuartxchange, 2013)

 

 

Background of the Study

Many human infections are caused by either bacteria or viruses. Bacteria are tiny

single-celled organisms, thought by some researchers to be related to plants. They are

among the most successful life forms on the planet, and range in habitat from ice slopes

to deserts. Bacteria can be beneficial – for instance, gut bacteria help us to digest food

– but some are responsible for a range of infections. These disease-causing varieties

are called pathogenic bacteria.

Many bacterial infections can be treated successfully with appropriate antibiotics,

although antibiotic-resistant strains are beginning to emerge. Bacterial infections can be

caused by a wide range of bacteria, resulting in mild to life-threatening illnesses (such

as bacterial meningitis) that require immediate intervention. According to Howard

(1994), in the United States, bacterial infections are a leading cause of death in children

and the elderly. Common bacterial infections include pneumonia, ear infections,

diarrhea, urinary tract infections, and skin disorders. (Life Extension, 2013)

The fight against bacterial infection represents one of the high points of modern

medicine. The development of antibiotics in the 1940s offered physicians a powerful tool

against bacterial infections that has saved the lives of millions of people. However,

because of the widespread and sometimes inappropriate use of antibiotics, strains of

bacteria have begun to emerge. These strains are more antibiotic-resistant. These new,

stronger bacteria pose a significant threat to general health and welfare – and a

challenge to researchers. (Life Extension, 2013)

Development of microbial resistance to the available antibiotics have lead

researcher to investigate the antibacterial activity of medicinal plants. Today,

researchers continue to extend their efforts in getting scientific information on plants

naturally occurring here in the Philippines. This would be of great importance to the

solutions of many critical health problems as a way of giving much attention, to the

utilization of drug from the Philippine medicinal plants than from chemicals in order to

provide better and safer medication. Many of the country’s medicinal plants contains

different constituents and therapeutic properties. One of these naturally occurring plant

is the Alambangbang (Bauhinia malabarica) from Family Fabaceae. According to Gana,

Bauihinia malabarica contains tannins.

Tannins are polyphenolic substances found in leaves, bark, wood and bound to

proteins that form insoluble or soluble tannin – protein complex. PHARMACOLOGICAL

ACTIVITY OF TANNINS.

The plant material to be used in this study will be gathered from Laguna

Province.

STATEMENT OF THE PROBLEM

The study will deal with the determination of antibacterial activity of tannins from

the leaves of Bauhinia malarabica.

Specifically, it sought to answer the following questions:

1. What constituents are present in the extract of Bauhinia malarabica?

2. What are the physical and chemical tests to confirm the presence of tannin?

3. What will be the procedure in purifying the tannin extract?

4. What type of microbiological screening will be used to test the antibacterial

property of the plant extract?

5. Will tannin extract be effective as antibacterial agent?

SCOPE AND LIMITATION OF THE STUDY

This study focused mainly on the determination of the anti-bacterial property of

the tannin content of Bauhinia Malabarica leaves.

Collection and preparation of materials is done for preliminary test of tannins

Is conducted to prove its presence.

Qualitative chemical, physical analysis, antibacterial screening using filter paper

Disc method against Escherichia coli is use to determine the presence and

activity of the tannin extract, and comparing the antibacterial activity of the tannin

extract with ampicillin trihydrate.

Significance of the study

The study of the anti-bacterial property of Alambangbang leaves will benefit the

following:

For educators and students - the study can provide topics for classroom discussions

and serve as reference for the other subjects related to the study about plants.

For future researchers - this research can provide basis and references for future

studies related to Bauhinia Malabarica

Researchers of this Study - The researchers, being the main participants, will gain

knowledge and experience to improve their skills. This research is a major requirement

to complete the Bachelor of Science in Pharmacy program of the St.Jude College-

Manila.

For Health Sectors – they the advocate of promoting heaths thus this study will benefit

certain patients if the drug itself is cheaper and safer.

For the manufacturer - for further study and investigate in its quality in order to

formulate and designed products with an economical price.

DEFINITION OF TERMS

ANTIBACTERIAL.A substances that kills bacteria or inhibits their growth or

replication. ( Taber,1977 )

AUTOCLAVE.A laboratory instrument that sterilizes microbiological materials

by means of steam under pressure. ( Webster,1990 )

CONTINUOUS EXTRACTION.A process of removing a solvent which has been

vaporized and which is condensed and use over and over again for the purpose of

soluble material origin substance. ( Webster 1990 )

EXTRACTIVE. Having the nature of an extract, the product of extraction

process. ( Taber,1977 )

EXTRACT. An essence or concentrate extract, the product of extraction by

pressing distilling or obtained by dissolving a drug in some solvent, as ether or alcohol,

and then evaporating the preparation. ( Webster,1990 )

INCUBATION PERIOD. An elapsed time between the exposures to an infection

and the appearance of disease symptoms, or the time period during which

microorganisms inoculated into a medium are allowed to grow. ( Palezar, 1993 )

INFUSION.A Liquid preparations by sleeping the medicinal herb or part of the

plant in boiling water for atleast 15 minutes, also known as teas. ( de Padua, 1977 )

INOCULATION. An artificial introduction of micro organisms or other substance

into the body or into a culture medium.( Palezar,1993 )

MACERATION. A process in which a drug is sleeped in a liquid and continued

application of herb until the tissue becomes softened and soluble matter contained in

the cells has dissolved. ( Tyler, 1998)

MARC. An undissolved portion of the drug that remains after the contraction

process. ( Tyler, 1998 )

MENSTRUM. An excretion removes only those substances that can be

dissolved in the liquid or liquid mixture. ( Tyler, 1998 )

SOXHLET. An apparatus generally used for extraction with volatile solvents

where small quantities of a drug are extracted, as in quantitative work. ( Tyler, 1998 )

TANNINS. A complex substances usually occurs as mixtures of polyphenols

that are very difficult to separate since they do not crystallize

CHAPTER 2

THE REVIEW OF RELATED LITERATURE

The review of related literature and studies will give some background of the

study conducted. Its taxonomic classification, botanical description and uses, chemical

composition and other pharmacological studies of the plant material, plants with similar

pharmacological property have also included.

This summary of a relevant facts about the Alambangbang plants, tannin and

anti-bacterial activities.

Taxonomic Classification and Botanical Description of the Plants

Taxonomic Classification: Alambangbang belongs to the genus Bauhinia

species Malabarica with a binomial name of Bauhinia malabarica is synymous to

Bauhinia tomentosa Blanco, Bauhinia purpurea Vidal, Piliostigma acidum Benth and

Piliogtima Malabaricum (Roxb) Benth. Its scientific name is Bauhinia malabarica Roxb.

Of the family Fabaceae. Being one of the largest family found in all parts of the world, it

consists of 500 genera and 12,000 species, 91 genera and about 270 species are

grown in the Philippines. It also occurs in India to Indochina, Java and Timor.

(Quisumbing, 1978).

Botanical Descriptin: This is a small sized but stocky tree reaching a height 8 to 10

meters with yellowish-brown, checked bark. The branches are really rebranched,

forming a dense crown, the ultimate being one smooth. The leaves broader than long

are 5 to 10 centimeters in length, heart shape at the base, and deeply notched at the

apex. The flowers are white and rather large. The pods are long, narrow and flattered

being 20 to 30 centimeters by 1.8 to 2.5 centimeters. (Quisumbing, 1978)

Small bushy tree, twigs and leaf stalks pinkish brown, blade soar. Flowers less

than 1 inch wide, creamish white, 10 stamens, calyx not split. Leaves small, soar edible.

Tree very fire resistant. (Steiner, 1960)

Distribution and Habitat. Alambangbang has a wide distribution namely;

English (purple bauhinia,orchid tree,camel's foot tree,butterfly tree,geranium tree); Hindi

(kota,raktakanchan,khairwal,karar,kanchan); Malay (tapak kuda); Nepali (tanki);

Spanish (pie de cabra); Thai (sieowaan,sieo dok daeng); Trade name

(kachan,karar,khairwal);

Bauhinia Malabarica is a genus of more than 200 species namely; The genus

was named after the Bauhin brothers, Swiss-French botanists. The species share the

'butterfly' configuration of the leaves. Alambangbang is an Ilongo word for butterfly. It is

very common in open, dry slopes, in regions with long dry seasons in Luzon (Ilocos

Norte to Laguna). Also occurs in India to Indo-China, Java and Timor. (Stuartxchange)

Native in Thailand, Australia, India, Indonesia, Malaysia, Myanmar and the

Philippines. In its natural habitat in Australia, China, India, Philippines, the tree is

deciduous. Flowering from September to November occurring when the plant in

leafless. Tree starts flowering at a very early age of 2-3 years and yields viable seed.

The seed ripens between February and May, with a tendency to be later in the West

than in the East. The seeds disperse from the pods and germinate on sites with

favourable light and moisture conditions, while in unfavourable niches the radical dries

up or is destroyed by birds. The pods should be collected when they are brown before

the dehisce. The seeds are extracted by drying the pods in the sun to release seeds.

Bauhinia Malabarica is a plant of tropical and subtropical climates usually found

in most types of vegetation ranging from evergreen lowland rain forest to mountain

forest and savanna, scrub and dry deciduous forest to swamp foreston various soils. It

occurs at lower elevations especially frequent along the valleys in its native habitat. It

demands plenty of light and requires good drainage. Severe frost kills the leaves of

seedlings and saplings, but they recover during summer. The species is frost-hardy but

least drought-hardy compared to other species of Bauhinia. The tree actually flower

best on dry soils.

Propagation and Cultivation of the Plant. This is propagated by seeds,

cuttings, or by marcotting. Propagation by seeds is either direct seeding, or raised at the

nursery readied for outplanting in the field. (Food from the Wilderness, Nov. 2003)

Chemical Composition of Alambangbang. Bark contains a tannin, 9.5 %.

• From the methanol extract of leaves: 6,8-di-C-methylkaempferol 3-methy ether,

kaempferol, afzelin, quercetin, isoquercitrin, quercitrin, and hyperoside. source

• Oil extracted from the seeds was 16%, with linolenic 0.81%, linoleic, 47.26%, oleic

15.26%, stearic 19.29%, palmitic 17.18%, myristic 0.02%.(Stuartxchange)

Harvesting.

Beneficial Aspects.

Tannin. Tannin compounds are widely distributed in many species of plants, where

they play a role in protection from predation, and perhaps also as pesticides, and in

plant growth regulation. The astringency from the tannin is what causes the dry and

pucker feeling in the mouth following the consumption of unripened fruits.

Tannin are mainly physically located in the vacuoles or surface of plants. The

storage sites keep tannins active against plant predators, but also keep some tannins

from affecting plant metabolism while the plant tissue is alive, it is only after cell

breakdown and death that the tannins are active in metabolic effects. Tannins are

classified as ergastric substances, i.e.., non-protoplasm materials found in cells.

The most abundant polyphenols are the condensed tannins, found in virtually all

families of plants, and comprising up tp 50% of the dry weight of leaves.

Plant Source of Tannins. Tannins are found in leaf, bud, seed, root, and stem

tissues. An example of the location of the tannins in stem tissue is that they are often

found in the growth areas of tress, such as the secondary phloem and xylem and the

layer between the cortex and epidermis. Tannins may help regulate the growth of these

tissues.

The best known families of which all species tested contain tannin are:

Aceraceae , Actinidiaceae, Anacardiaceae, Bixaceae, Burseraceae, Combretaceae,

Dipterocarpapaceae, Ericaceae, Grossulariaceae, Myricaceae for dicot and Najadaceae

and Typaceae in Monocot. To the family of the oak, Fagaceae, 73% of the species

tested ( N=22) contain tannin. For those of acacias, Mimosaceae, only 39% of the

species tested (N=28) contain tannin, among Solanaceae rate drops to 6% and 4% for

the Asteraceae. Some families like the Boraginaceae, Cucurbitaceae, Papaveraceae

contanin no tannin-rich species.

Nutritional and Medicinal Uses of Tannins Foods rich in tannin can be used in the

treatment of the HFE hereditary hemochromatitis, a hereditary disease characterized by

excessive absorption of dietary iron, resulting in a pathological increase in total body

iron stores.

Recent studies have demonstrated that products containing chestnut tannins

included at low dosage (0.15-0.2%) in the diet can be beneficial. Some studies suggest

that chestnut tannins have been shown to have positive effects on silage quality in the

round bale silages, in particular reducing NPNs ( non-protein nitrogen ) in the lowest

wilting level.

It is believed that tannins isolated from the stem bark Myracrodruon urundeuva

may have nuero-protective functions capable of reversing 6-hydroxydopamine-induced

toxicity. The plants has shown promise as a potential therapeutic agent, which may be

beneficial in patients with neurological disease. Souza et.al discovered that the tannins

isolated from the stem bark also have anti-inflammatory and anti ulcer activity in

rodents, showing a strong anti oxidant property with possible therapeutic applications.

Improved fermenting ability of soya meal nitrogen in the rumen has also been

reported by F. Mathieu and J.P Jouany (1993). Studies by S. Gonzales et. al (2002) on

in vitro ammonia release and dry matter degradation of soybean meal comparing three

different types of tannins ( quebracho, acacia and chestnut ) demonstrated that chestnut

tannins are more efficient in protecting soybean meal from in vitro degradation by rumen

bacteria.

It is found out that the root, bark and wood of the Pterocarpus Indicus have

components that are said to have immune enchancing and purported use for the

prevention and treatment of asthma. The Polyphenolic Antiplasmin constituents that

were found on its bark have showed carcinostatic effects on ascetic mice with Erlich

carcinoma. In soutn Africa, ethno-veterinary medicine was widely used in the

Antibacterial activity of Pterocarpus indicus, ( Fitoterapia 74, 2003)

Tannins may also be effective in protecting the kidneys. When incubated with red

grape juice and red wines with a high content of condensed tannins, the poliovirus,

herpes simplex virus and various enteric viruses are inactivated.

Tannins have shown potential antiviral (Lin et. al.., 2004), antibacterial (Akiyama

et. al.., 2001) and antioxidant activity evaluated for their antioxidant ability with a 1,1-

diphenyl-2-picrylhydrazyl (DPPH) radical generating system. Tannins have also been

studied for their potential effects against cancer through different mechanism

(Bhagavathi, 1999)

It has also been reported to have numerous other biological or pharmacological

properties, including antimicrobial (see balm), growth suppression (in rats), nonspecific

CNS depression (in mice) cariostatis (in harmsters), hypoglycemic (in humans),

antifertility (in males), and an antiamyloidogenic activity in vitro through destabilizing

Alzheimer B-amyloid fibrils. Earlier studies in mice also showed that tannin acid inhibits

hyaluronidase enzyme and may have potential in manging subcutaneous poisoning

resulting for snakebites. (John Wiley, 2010)

Extraction of tannins. There is no single protocol for extracting tannins from all plants

material. The procedures used for tannins are widely variable. It may be that acetone in

the extraction sovent increase the total yield by inhibiting interactions between tannins

and proteins during exraction or even by breaking hydrogen bonds between tannin

complexes.

Lyophiilization ids thought to be the gentlest method of preservation and

lyophilized leaves may be equivalent to fresh leaves (Martin and Martin, 1983).

However, diminished extract bility of tannin after lyophilization has been noted in some

cases (Price et. al.., 1979). Dying the tissue at ambient or at elevated temperature is

more convenient than lyophilisation, although little tannin can be extracted from

samples dried at elevated temperature (Bale-Smith, 1975; Price et. al.., 1979). Lower

drying is preferable. ( Swain, 1979), but even drying at room temperature may alter the

chemical nature of tannin (Butler, 1982.)

Three solvents are commonly used to extract tannin from plant sample: boiling

aqueous methanol, aqueous acetone, or acidic methanol, Boiling aqueous methanol is

thought to be the most effective solvent for condensed tannin (Bate-Smith, 1975), but

the recovery of tannin is estimated as low as 30% for some tissues (Bate-smith 1973a;

Swain, 1979). Since aromatic ester (depside)bonds are hydrolysed by aqueous

alcohols., they are thought to be unsuitable for extraction of hydrolysable (Haslam et.

al..,1961; Swain 1979).

Aqueous acetone is routinely used to extract hydrolysable tannin (Jones et. al..,

1979; Foo and Porter, 1980), but no quantitative estimates of recovery are available.

Some authors believe condensed tannin is extracted quite efficiently with

aqueous acetone (Feeny and Bostock, 1968; Fitcher et. al.., 1977: Lane and Schuster,

1981), but others have found that condensed tannin is recovered in low yields when

aqueous acetone is employed (Stafford and Cheng, 1980: Martin and Martin, 1984).

Acidic methanol is the best solvent for extracting the condensed tannin for some

varieties of sorghum (Price et.al.., 1978).

Characterization of Tannins. The United States Pharmacopeia provides quality criteria

for the characterization of tannin. Characterization procedures are carried out using

different physical and chemical tests and instruments methods. Solubility is measured

dissolving 10grams of substance in 20mL in boiling water and diluting it in enough water

to make a 200mL.

Bacterial infections

Types with pathophysiology

Sign and Symptoms and Complication

Treatment of Bacterial infection

Related Studies

Studies on other plants parts of Alambangbang. Antioxidant: In a DPPH assay,

leaf extracts exhibited very potent antioxidant activity, compared with pure catechins

used as positive controls.

Flavonoids / Antioxidant: Study of ethyl acetate extract of leaves isolated 2 active

compounds: quercetin-3-O-rutinoside and quercetin. Compound 2 had higher

antioxidant activity while compound 1 had lower activity than L-ascorbic acid.

 Reproductive Benefits: Study of aqueous and ethanol extracts of dried leaves on

pregnant Wistar rats showed no evidence of maternal or fetal toxicities and the aqueous

extract promoted increased implantation and decreased post-implantation loss in

pregnant rats.

SeedsNutritional Study / Potential Nutrient Source: Protein content was 33.09%,

comparing fairly with soya beans (Glycine max) and groundnuts (Arachis hypogaea).

Seeds also yield 21.45% soluble carbohydrates, comparing favorably with soybean

20.7% and peanut 24.6%. It also yields 3.25% fiber. The hydrogen cyanide content was

0.32 mg/100g; chronic exposure has been reported to cause neurological, respiratory,

cardiovascular and thyroid debilities. Study concludes BM seeds are rich in nutrient and

low in anti-nutritional compounds. If popularized, the plant may serve as a

complimentary source of essential nutrients to man and livestock provided toxicants are

removed. (Herbal-medicine.net76).

Antibacterial activity of tannin.

Plant that contain tannin.

Studies of antibacterial activity.