Flavonoids, Alkaloids and Tannins of Three Varieties of Horse Radish (Moringa Oleifera

Lam.) Extracts: Potential Components of Toothpastes

Jhoseph B. Carranza,Precious Gem L. Molina,John Joseph R. Ortañez, Lorcelie B. Taclan*,

Vicky C. Mergal and Orlex Yllano

Adventist University of the Philippines

Puting Kahoy, Silang, Cavite

lbtaclan@yahoo.com

Moringa oleifera Lam is a very common plant in tropical countries such as the

Philippines. Recently, the Philippine Congress approved it as the national vegetable because of its

various health benefits. The study generally identified the secondary compounds of the three

varieties namely; Native, Yard Long, and Chinese through phytochemical analysis. Specifically,

to establish the secondary compounds of the three varieties that would be potential components of

toothpaste. Gathering of samples of the three varieties was done at the university campus. Each

variety was dried using the Multi commodity heat pump dryer to preserve their nutritional

contents before crude extraction that was done at the Department of Science and Technology-

Industrial Technology Development Institute (DOST-ITDI) using 95% ethanol. Each extract was

subjected to phytochemical analysis using standard laboratory procedures. Results revealed that

the extracts contain flavonoids, alkaloids and tannins that are known to have antimicrobial and

anti-inflammatory properties, thus, these Moringa varieties have potential natural components in

the manufacture of toothpastes.

Keywords: Moringa oleifera L., flavanoids, alkaloids, tannins

Growing up in a tropical country, people have the tendency not to notice the beauty of the

vegetation that surrounds them. More often than not, the plants that are easily seen in our

backyard are taken for granted. Why say this? It is because the researchers are also guilty of it.

For example, Moringa oleifera Lam. or commonly called as malunggay in the Philippines, the

plant that is present in every Pinoy’s food, backyard, or sometimes in an abandoned lot. Not only

Moringa oleifera Lam. is easily seen in the Philippines, the country has the most researched

proven variety of the commodity. According to Nelly Favis-Villafuerte (2015), there are 13

different species of moringa plant and the best-known species and the most widely cultivated is

this specie native to the Philippines. The leaves, flowers, seeds, pods, roots, bark, gum, and the

seed oil from the malunggay plant are continually being subjected to intensive research and

development programs because the various constituents of the Moringa oleifera Lam. are known

to have, among other properties, anti-diabetic, anti-hypertensive, anti-inflammatory,

anti-microbial, anti-viral, anti-parasitic, anti-tumor and anti-aging activities. Though the plant is

native to the Philippines, it was also entitled as one of the world’s most useful plant (Marro,

2004).

The human’s oral cavity has many microorganisms inside, be it anaerobes or aerobic

bacteria. In fact, over 700 different kinds of bacteria have been detected in the human oral cavity.

The normal number of strains that can be found on a human oral cavity is 34 to 72 varieties only.

These are not harmful bacteria (Woudstra, 2015). Every part of the oral cavity has a different

concentration of microorganism. A study entitled Aerobic and Anaerobic bacteria in subgingival

and supragingival plaques of adult patients with periodontal disease compared the bacteria

present in plaques supragingival and subgingival. A total of 42 bacterial strains were isolated

from subgingival plaques, which of 24 (51.1%) belonged to 7 anaerobic species and 18 (42.9%)

to 12 aerobic species. There were more aerobic (33/53; 62.3%) than anaerobic bacteria (20/53;

37.7%) in supragingival plaques (Daniluk, 2006). These bacteria help in the first stage of food

digestion which is the breaking down or chewing of food into smaller pieces. The bacteria are

with the saliva that initiates the digestion and aid in chewing the food. The food then is made into

smaller, soft mass called bolus (YGOY Health Community, 2015). One of the most harmful

bacteria that can be found in the oral cavity is the Streptococcus mutans. It lives inside the mouth

and feeds on the sugars and starches that a person eats. That alone wouldn’t be so bad, but as a

by-product of its ravenous appetite, it produces enamel-eroding acids, which make Streptococcus

mutans the main cause of tooth decay in humans (Woudstra, 2015). Streptococcus mutans is an

indigenous micro flora of the oral cavity. It cannot be completely eliminated from the mouth and

the more it stays in the oral cavity, feeding on the starch and sugar that the person ingests, the

more they become in number that will be very bad and will result in greater probability of having

dental caries. Once a person gets a strain of oral bacteria, the person would not likely to get rid of

it but the good news is that people can control the bacteria living inside their mouth with good

oral hygiene. A study published in the Journal of Periodontology (JOP), the official publication of

the American Academy of Periodontology (AAP) demonstrates that flossing when made a routine

can actually help in reducing the amount of gum disease-causing bacteria that are found in the

oral cavity, thus giving people a better oral health. (American Academy of Periodontology, 2016)

Objectives of the Study

General Objective

To identify the secondary compounds of three varieties of Moringa oleifera Lam.

extracts namely; Native, Chinese, and Yard Long malunggay using phytochemical analysis.

Specific Objectives

1. To identify the appropriate solvent in the extraction of the secondary compounds of

native, yard long, and Chinese malunggay;

2. To identify the secondary compounds of the three varieties of malunggay that would be

potential components of toothpaste.

Review of Literature

Moringa oleifera Lam.

Malunggay in the Philippines is well known for its nutritional value as herbal medicine

(Fuglie, 2000). This plant is well cultivated in areas with tropical climate such as the Philippines,

India and Africa. This plant can grow to as high as 9meters with erect white trunks. The

compound leaf has about 3 to 9 leaflets. The plant has white fragrant flowers that produceslong

pods with 3-angled winged seeds (Medical Health Guide, 2011).

The Horse radish plant scientific classification:

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Brassicales

Family: Moringaceae

Genus: Moringa

Species: Moringa oleifera Lam.

Different solvents used in the extraction of phenolic content of plants

Three genotypes of horseradish roots were collected in (latitude 57° 03’ N, longitude 22°

91’ E) during the period from September to November, 2011. For analyses, the average sample of

300 grams was collected by the researcher; this study took 3 unknown genome of roots. Each

genome of fresh roots were washed, peeled and homogenized (for 5 minutes). All samples of one

type of horseradish were homogenized together in order to obtain representative sample.

Figure 1 Total phenolic content of horseradish extracts

Reading of the Total Phenolic Content of horseradish extracts showed differences depending

on solvent used, namely: n-hexane (HE), ethyl acetate (EA), diethyl ether (DI), 2-propanol (PR),

acetone (AC), ethanol 95% (ET), ethanol/water/acetic acid (80/20/1/v/v/v) (EWA), and

ethanol/wataer (80/20 v/v) (EW). As the best solvents ethanol and ethanol / water solutions can

be chosen (Tomsone, 2012).

Effect of ethanol/water solvent on phenolic contents

A study revealed that phenolic compounds extracts were significantly dependent on the

concentration of ethanol/water solvent. The study revealed the highest phenolic content is

extracted when ethanol/water was used as a solvent compared to other solvents in the study

(Chunli, 2015).

Secondary Components of Moringa oleifera Lam. as an Anti-microbial

Flavonoids and alkaloids that are found in some plants is said to have an antiviral and

antimicrobial activities. A study done by the Faculty of Pharmacy, Department of Pharmaceutical

Microbiology, Gazi University, Ankara, Turkey proved that this was true. They tested the antiviral

activity of the compound by testing it against DNA virus herpes simplex type 1 and RNA virus

influenza (type-3). The Antibacterial activity was assessed against the following bacteria and their

isolated strains: Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella

pneumoniae, Acinetobacter baumanni, Staphyloccocus aureus, Enterococus faecalis, and Bacillus

subtilis (Ozcelik, 2011). Adding to the wonders of flavonoids, a study by Salvatore Chirumbulo

stated that flavonoids has anti-inflammatory agents thus making it a great potential to be used as a

main ingredient in an anti-inflammatory drug (Chirumbulo, 2010). To prove the antibacterial

effect of Alkaloid, Shachi Singh et al did a study on several gram positive and gram negative

bacterial strains and arrived to a conclusion that the antibacterial test conducted revealed that

alkaloids have antibacterial property (Shachi et al, 2011). Phenolic compound on each plant

differs, therefore. it is hard to develop a standard extraction procedure suitable for the extraction

of all plant phenols. But, for the extraction of Moringa oleifera Lam., the use of ethanol and

ethanol/water (80/20 v/v) are the best solvents to use (Tomsone 2012).

Antibacterial Potential of Moringa oleifera Lam.

This present study is similar to that of Caurez (2012), the researcher evaluated the

antibacterial potential of Moringa oleifera Lam. root extracts in gram-positive (Staphylococcus

aureus and Bacillus subtilis) and gram-negative (Eschirichia coli and Salmonella typhii) bacteria.

The aqueous (distilled water) and alcohol (95% ethyl alcohol) root extracts of Moringa oleifera

Lam. exhibited antibacterial activity against S. Aureous and S. typhii. On table 1 is the

antibacterial acvitiy of Moringa Oleifera Lam. leaf juice extracts against some human pathogenic

bacteria.

Table 1 Antibacterial acvitiy of Moringa Oleifera Lam. leaf juice extracts against some human pathogenic bacteria (Caurez 2012)

Bacteria

Zone of Inhibition(mm)

Fresh leaf juice Aqueous extractsa

Aqueous extractsa

Ethanol Extractsa Positive Control

Tetracycline

Liquidb Powdera dissolved in DMSO

Fresh leaves Dried leaves Fresh

leaves Dried

Leaves

Cold Hot Cold Hot

Gra

m N

egat

ive

Shigella shinga 20.2±0.04 36.2± 0.08 7.75±0.56 + + + 17.5±0.34 + 12.20±0.13

Pseudomonas aeruginosa 17.00±0.66 39.60±0.49 15.00±034 + + + 21.21±0.05 + 18.30±0.12

Shigela sonnei 25.1±0.12 33.5±0.12 13.45±0.04 + + + 21.50±0.08 + 14.16±0.23

Pseudomonas spp. 25.2±0.04 42.3±0.16 27.5±0.21 + + + 21.25±0.1.3 + 20.16±0.19

Gra

m P

ositi

ve

Staphylococcus aureus 15.23±0.05 36.4±0.08 12.0±0.12 + + + + + 12.76±0.02

Bacillus cereus 22.4±0.28 29.25±0.2 8.00±0.42 + + + 16.25±0.04 + 9.34±1.3

Streptococcus-B-haemolytica 18.0±0.04 35.15±0.12 10.75±0.24 + + + + + 8.33±2.01

Bacillus subtilis 21.6±0.04 33.75±0.2 17.25±0.14 + + + 20.23±0.56 + 7.25±0.08

Sarcina lutea 18.1±0.04 34.4±0.44 8.50±0.09 + + + 19.50±0.21 + 8.66±0.13

Bacillus megaterium (Entero) 19.0±0.04 39.25±0.2 14.75±0.04 + + + 20.50±0.04 + 20.16±0.43

Values are presented as mean ± S.E. of triplicate experiments. + = Growth a Diameter of inhibition zone

including diameter of disc 6 mm (tested at a volume of 10 μl/disc at a concentration of 1175μg disc-1). b Diameter of inhibition zone including diameter of disc 6 mm (tested at a volume of 10 μl disc-1). DMSO = Dimethylsulfoxide

In the following aspects, the study used the extracts of Moringa oleifera Lam. leaves and the

organism tested is Streptococcus mutans. Philippine Council for Health Research and Development

published an article entitled “Moringa Potential Ingredient for Mouthwash/toothpaste”, the article

expounded that the study was conducted by researchers from Our Lady of Fatima University. In the study,

the result of the microbial in vitro test performed by the University of the Philippines Los Banos-National

Institute of Molecular Biology and Biotechnology (BIOTECH_UPLB) showed that juice of Moringa oleifera Lam. leaves inhibited the growth of Streptococcus mutans. Thus, the result of the present study

provided a framework for the researchers’ future study in the development of a Moringa oleifera Lam.

toothpaste for the inhibition of Streptococcus mutans in the oral cavity.

Components of a Basic Toothpaste

Dental Health Foundation in Ireland made a detailed and interesting article about the ingredients

of a basic toothpaste which is composed of abrasives, detergent, binding agents, humectants, flavoring

sweetening and coloring agents, preservatives, water, fluoride, and other therapeutic agents. Abrasive. Abrasive are the cleaning and polishing agents in a toothpaste and account for about a

third of the toothpaste by weight. Most abrasives are chalk or silica based. Examples are dicalcium

phosphate, sodium metaphosphate, calcium carbonate, silica, zirconium silicate or calcium pyrophosphate.

Abrasives differ in strength. An international standard defines a test paste against which toothpaste

abrasivity can be assessed. The unit of measurement is known as the Relative Dentine Abrasivity (RDA)

or the Relative Enamel Abrasivity (REA)

Detergent. Detergent (1-2%) foam and loosen plaque and other debris from the tooth surface.

Principal examples are sodium lauryl sulphate and sodium N-lauroyl sarcosinate.

Binding agents. Binding agents (1%), these agents prevent separation of solid and liquid

ingredients during storage. They are usually derived from celullose, sodium carboxy-methyl cellulose

being the most commonly used. Carrageenans (seaweed derived), xantham gums and alginates are also

used.

Humectants. Humectants (10-30%) acts to retain moisture and prevent the toothpaste from

hardening on exposure to air. Glycerol, sorbitol and propylene glycol are commonly used. Glycerol and

sorbitol also sweeten the toothpaste, though this is not their main function.

Flavoring, sweetening and coloring agents. (1-5%) Peppermint, spearmint, cinnamon,

wintergreen and menthol are among the many different flavoring used. While rare, mucosal irritations

from toothpaste (i.e., ulcerations, gingivitis, angular cheilitis, perioral dermatitis) are usually linked to

flavourings or preservatives they contain.

Preservatives. (0.05-0.5%) Alcohols, benzoates, formaldehyde and dichlorinated phenols are

added to prevent bacterial growth on the organic binders and humectants.

Fluoride and other therapeutic agents. The majority of toothpastes combine the caries

protection of fluoride with other therapeutic agents to control plaque, tartar and gum disease. The

inclusion of antibacterial agents can help individuals improve their plaque control. Mostly toothpastes

include triclosan, which has been shown to offer a clinically useful improvement in gum health. Other

pastes specially target “tartar” (hardened plaque) and use phyrosphosphate to inhibit the mineralisation of

dental plaque and hence the buildup of tartar (calculus). Toothpastes with desensitising agents are also

available for sensitive teeth. A study was conducted using an in vitro test of the effectiveness of

malunggay using disc diffusion and minimum inhibitory concentration (MIC) determination method

against human pathogenic bacteria. The study tested Shigella shinga, Pseudomonas aeruginosa, Shigella

sonnei, Pseudomonas spp, staphylococcus aureus, Bacillus cereus, Streptococcus-B- haemolytica, bacilus subtilis, sarcina lutea, Bacillus megaterium. The tests done using fresh leaf juice, powder from fresh leaf

juice, and cold-water extracts of fresh leaf exhibited inhibitory effect against all the tested Gram-negative

bacteria and Gram-positive bacteria and that includes Streptococcus-B-. However, the test using ethanol

extracts of fresh leaves gave a positive result on both Gram-negative and Gram-positive bacteria except in

S. Aureus and Streptococcus-B-haemolytica. The consequences of this investigation suggest that the

extracts and juice of M. oleifera Lam. can be used to discover antibacterial agent for developing new

pharmaceuticals to control studied human pathogenic bacteria responsible for severe illness” (Rhaman

2009). The related studies presented here were selected on the basis of their significance in promoting

directions for this present study. Streptococcus mutans is a good factor in the initiation of caries formation

(Simon 2007). The researcher expounded two pointers why Streptococcus mutans is a potent initiator of

caries. “Firstly, S. mutans is an anaerobic bacterium known to produce lactic acid as part of its

metabolism. Secondly, there is the ability of S. mutans to bind to tooth surfaces in the presence of sucrose

by the formation of water-insoluble glucans, a polysaccharide that aids in binding the bacterium to the

tooth” (Simon 2007). Also, unique to other species of microorganisms present in the oral cavity,

Streptococcus mutans does not slow down its metabolism even in a low pH environment. The effect of

flavonoids, alkaloids and tannins was established to have antimicrobial and anti-inflammatory effect by

previous studies, thus making it a potential component of toothpaste to reduce bacterial growth and

induce anti-inflammatory action.

Materials and Methods

Collection and preparation of Moringa oleifera Lam. for drying

The researchers gathered three kilograms of fresh Moringa oleifera Lam. for each variety to have

a yield of 300 grams dried of each. The researchers needed to have a minimum of 300 grams dried for

each variety in able to be tested for phytochemical analysis and antimicrobial susceptibility test. Weighing

scale was used to have an accurate measurement and to compute how much dried Moringa oleifera Lam.

the researcher can have in every certain weight of fresh Moringa oleifera Lam. that was subjected to

drying. Multi Commodity Heat Pump Dryer (MCHD) was used as the means of drying each variety; this

facility dehydrated the samples at an ideal drying condition which is 50°C. This equipment ensures the

embedded nutritional values, as well as the color of the samples will be retained after drying (Taclan,

2012). The researchers made sure that they got the desired dryness of all the varieties by weighing the

fresh varieties before drying. After 5h of drying, the researchers weigh the dried varieties and subjected

the dried varieties for another hour. After an hour, each variety was weighed again and when the present

weigh matched the weigh an hour before, it is an assurance that all varieties are dried enough and ready

for the next procedure. Heavy duty grinder was used to reduce the size of the dried varieties into smaller

particles or powdered form for the samples to be extracted.

Identification of the appropriate solvent in the extraction of secondary compounds

After preparing and having the needed weight of 300 grams each variety, the ethanolic crude

extraction of Moringa oleifera Lam. extracts, was done with the help of Department of Science and

Technology Industrial Technology Development Institute.

First, each powdered variety was soaked in the researcher’s chosen solvent which is 1.8 liters of

95% ethanol for 48 hrs. The use of rotary evaporator device was used after 48h of soaking to finally

extract the needed solution for the phytochemical analysis. There were still traces of the solvent used in

the crude extracts thus the use of 60°C water bath was needed to vaporize the remaining solvent as

another purification process.

Crude Extraction of Malunggay Leaves - Ground

Native Malunggay. The 300 grams of Native malunggay leaves produced 1.8 liters of

ethanolic extracts. Which was filtered and yielded 25 grams of semi solid crude extracts.

Yard Long Malunggay. The 300 grams of Yard Long malunggay leaves produced 1.8

liters of ethanolic extracts. Which was filtered and yielded 32 grams of semi solid crude extracts.

Chinese Malunggay. The 300 grams of Chinese malunggay leaves produced 1.8 liters of

ethanolic extracts. Which was filtered and yielded 35 grams of semi solid crude extracts.

Phytochemical analysis

The extracts of each variety were subjected to phytochemical analyses at the National

Institute of Molecular Biology and Biotechnology (BIOTECH) to identify their secondary

compounds which were Sterols, Triterpenes, Flavonoids, Alkaloids, Saponins, Glycosides, and

Tannins.

Bacterial Specimen

Only one microorganism strain was used in the investigation for antimicrobial assay

namely Streptococcus mutans. The microorganism was obtained from the PNCM Staff Philippine

National Collection of Microorganism. National Institute of Molecular Biology and

Biotechnology. University of the Philippines Los Baños College Laguna 4031 Philippines.

Antimicrobial Susceptibility Test

Procedure was done by PNCM Staff Philippine National Collection of Microorganism.

National Institute of Molecular Biology and Biotechnology (BIOECH). University of the

Philippines Los Baños College Laguna using 8mm diameter of cylinder cup.

Antimicrobial Susceptibility Test Interpretation

After 24 to 48h of incubation a ‘halo’ or ‘clearing’ around each disc will appear, this is

known as Zone of Inhibition. The plates will be inverted and a ruler will be used to measure the

diameter zone of inhibition, in millimeter. Results will be expressed in millimeter diameter zone

of inhibition. The size of the paper disc will be also recorded. A metric ruler will be placed across

the zone of inhibition, at the widest diameter, and the disk will be measured from one edge of the

zone to the other edge. The disc diameter will actually be part of that number. The results

acquired will be compared to the zone of inhibition of the standard antibiotic Phenol for bacterial

strain.

Figure 2

Flowchart of the Study

Collection and preparation for drying

of the three varieties of Moringa

oleifera Lam.

Preparation of Moringa

oleifera Lam. extract

Perform

Susceptibility test

on S. mutans

(Verification)

Phytochemical

Analysis

Analyses and

interpretation of

the results

Identification of extraction solvent

Results and Discussions

Concentration of 95% ethanol was the best solvent identified in the extraction of the three

varieties of malunggay

This study was focused on the identification of the secondary components of malunggay

compound. Thus, the researchers used ethanol as a solvent. Ethanol was proven to be the best

solvent for the extraction of secondary compounds of plants (Tomsone 2012).

Secondary compounds of three varieties of malunggay as potential components of

toothpaste

Table 2 shows the secondary components found in the three varieties of malunggay as

potential components of toothpaste. Native and yard long malunggay has an abundance (+++) of

tannins, this further explains that incorporating this on a toothpaste will give to it an inflammatory

agent reducing gingivitis. This result is congruent to the findings of Chirumbulo (2016). Out of

the three varieties considered, native malunggay has a moderate (++) amount of flavonoids.

Flavonoids was proven to have antibacterial and anti-inflammatory effect (Pietta 2000). All three

varieties of malunggay show traces (+) of alkaloids. Alkaloids have an antibacterial effect

(Kakhia 2012). The effect of flavonoids, alkaloids, and tannins was established by previous

studies, thus making it a potential component of a toothpaste to reduce bacterial growth and

induce anti-inflammatory action. (Table 2) Based on the result of the phytochemical analysis, it

was proven that Moringa oleifera Lam. extracts contains flavonoids, tannins and alkaloids that

made the three varieties of Moringa oleifera Lam. potential component of a toothpaste.

Table 2

Secondary components found in the three malunggay variety as potential components’ for a toothpaste

Sample Native Malunggay Chinese Malunggay Yard Long

Malunggay

Sterols (+++) (+) (+++)

Triterpenes (-) (++) (-)

Flavonoids (++) (+) (+)

Alkaloids (+) (+) (+)

Saponins (+) (++) (+++)

Glycosides (+) (+) (+)

Tannins (+++) (+) (+++)

Note: (+) Traces; (++) Moderate, (+++) Abundant (-) Absence of Constituents Reference:

Pharmacognosy, 15th edition, 2002, Trease and Evans

Present Constituents of Moringa oleifera Lam. Extracts

Sterols

Sterols, also known as steroid alcohols, are a class of chemicals that play multiple

important roles in the body. They have parts that can dissolve in fat-like molecules and parts that

can dissolve in water. The most widely known human sterol is cholesterol, which serves as a

precursor to steroid hormones and fat-soluble vitamins. Some people take plant sterols such as

vitamins A, D, E and K as supplements (Fox et al, 2015). Sterols, particularly cholesterol, are

integral to the stability of the cell membrane. The membrane is the outer covering of the cell,

analogous to the skin. Chemically, it is called lipid bilayer; lipids refer to fat-like molecules that

function to keep the environments inside and outside the cell separate, and bilayer means there

are two layers. The cholesterol embedded in the membrane maintains flexibility, fluidity, and

providing stability to the membrane over a range of temperatures. Sterols work to reduce the

absorption of cholesterol in the gut so more is lost in the feces. This in turn helps to lower total

cholesterol and LDL-cholesterol (bad cholesterol) in the blood (Main, 2015). A 2003 study in the

"American Journal of Clinical Nutrition" showed that sterols may interfere with the absorption of

excess cholesterol from the diet and therefore may be good for the heart and blood vessels.

Though potential to many health benefits, long-term effects and side effects of taking sterols still

need to be established. Consultation to a physician is highly recommended if planning to take

sterols (Fox et al, 2015).

Triterpenes

Originally synthesized by plants as metabolites, and are abundantly present in the plant

kingdom in the form of free acids or aglycones (Esser, 1999), triterpenes are synthesized via the

mevalonate pathway and are not regarded as essential for normal growth and development, and

although they do exist in plants in simple unmodified form, they often accumulate as conjugates

with carbohydrates and other macromolecules, most notably as triterpene glycosides. Triterpene

glycosides have important ecological and agronomic functions, contributing to pest and pathogen

resistance and to food quality in crop plants. They also have a wide range of commercial

applications in the food, cosmetics, pharmaceuticals, and industrial biotechnology sectors

(O’Maill et al, 2014). Though without scientific basis, in many Asian countries, herbal products

containing triterpenes are widely prescribed to prevent or treat a variety of diseases by the

traditional healers (Pharmar, 2013).

Flavonoids

Flavonoids are phenolic substances isolated from a wide range of vascular plants, with

over 8000 individual compounds known. They act in plants as antioxidants, antimicrobials,

photoreceptors, visual attractors, feeding repellants, and for light screening. Many studies have

suggested that flavonoids exhibit biological activities, including antiallergenic, antiviral,

anti-inflammatory, and vasodilating actions (Pietta 2000).

Alkaloids

Alkaloids are naturally occurring chemical compounds containing basic nitrogen atoms.

The name derives from the word alkaline and was used to describe any nitrogen-containing base.

Alkaloids are produced by a large variety of organisms, including bacteria, fungi, plants, and

animals and are part of the group of natural products (also called secondary metabolites). Many

alkaloids can be purified from crude extracts by acid - base extraction. Many alkaloids are toxic

to other organisms. They often have pharmacological effects and are used as medications, as

recreational drugs, or in entheogenic rituals (Kakhia, 2012). Antibiotic activities are common for

alkaloids and some are even used as antiseptics in medicine, e.g., berberine in ophthalmics and

sanguinarine in toothpastes, however, it is difficult to know the extent to which alkaloids give

antimicrobial protection in the plant (Margaret, 2013).

Saponins

Saponins are the second metabolites that are widely distributed in the plant kingdom. It

acts as a chemical barrier or shield in the plant defense system to counter pathogens and

herbivores (Cheok, 2014). Saponins are rich in pharmaceutical properties and recently many

studies focus on saponins' ability to increase immune responses, and possession of antibacterial,

antioxidant, anticancer, antidiabetic and anti-obesity properties (Cheok, 2014). A study proved

that 100% methanol fraction of saponin-rich extracts from guar meal exhibited antibacterial

activities against Staphylococcus aureus, Salmonella Typhimurium and Escherichia coli, however

the results showed 20% and 60% methanol fractions stimulated Lactobacillus spp. growth.

Aginoside saponins extracted from A. nigrum L. roots had significant antifungal activity.

Saponins isolated from seeds of Capsicum annum L. showed higher antimicrobial activity against

yeasts compared to common fungi. The n-butanol extracts of shallot basal plates and roots

exhibited antifungal activity against plant pathogenic fungi (Cheok, 2014).

Tannins

Tannins are considered as protectants against the growth of microorganisms such as

bacteria and fungi. These compounds affect microbial growth adversely and provide resistance to

the plants against them. Tannins act as redox buffers in plant cells, they protect the plant from the

deleterious effect of light and also impart antidessicant properties to the plant. A carbohydrate

core appears to play an important role in reducing the reactivity and solubility of tannins. It

enables them to be transported and stored in the plant without producing toxic effect (Pandey,

1993).

Glycosides

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.

Glycosides can be classified by the aglycone, glycone, or glycosidic bond. If the glycone portion

of a glycoside is glucose, then the researchers refer to the molecule as a glucoside. If the glycone

portion of the glycoside is fructose, then the researchers refer to the molecule as fructoside. If the

glycone portion of the glycoside is glururonic acid then the researchers refer to the glycoside as

a glucuronide. In the human, toxic substances are often bonded to glucuronic acid. The

glucuronic acid functions to increase the water solubility of the glycoside, which can be more

easily secreted. (http://www.nutriology.com/glycoside.html)

Comparison and Discussion of Different Solvents for Isolation of Phenolic Compounds

Phenolic content of plant extracts are affected by different factors such as, variety of

plant used, climate where the plant was taken, storage of the plant before and after extraction,

handling and processing during testing etc. Plants grown in different part of the globe was proven

to have different Total Phenolic Content. The recovery of polyphenols from plant materials is

influenced by the solubility of the phenolic compounds in the solvent used for the extraction

process. The solvents listed below are arranged in their unpolarity starting from the most unpolar.

Solvent polarity plays a key role in increasing phenolic solubility. It was proven that Total

Phenolic Count generally increased by increasing a polarity of solvents. Phenolic compounds on

each plant also differs, therefore, it is hard to develop a standard extraction procedure suitable for

the extraction of all plant phenols (Tomsone 2012).

Summary and Recommendations

Summary

The plant constituents analyzed were Sterols, Triterpenes, Flavonoids, Alkaloids,

Saponins, Glycosides, and Tannins. Native and Yard Long malunggay have a close presence of

components in general yet have a slight difference between Saponins. Yard long have an abundant

constituent while Native malunggay have only traces and Flavonoids; wherein Native malunggay

have moderate constituents while Yard long malunggay only have traces. Chinese malunggay

have the least presence of components in general. Even with different concentrations, all three

varieties have presence of flavonoids, tannins, and alkaloids, which are potential antimicrobial

and an anti-inflammatory agent. The crude extracts were tested for its anti-microbial property on

Streptococcus mutans through antimicrobial susceptibility test done at National Institute of

Molecular Biology and Biotechnology. All three varieties with their different concentrations

resulted negative to the said test. Based on the researcher’s gathering of related literatures,

ethanol is the best solvent in extracting phenol rich plants such as the Moringa oleifera Lam.

Conclusions

The study showed that: (1) Ethanol was appropriate for the extraction of the three varieties of

malunggay namely: Native, Yard-long and Chinese and; (2) based on the result of the

phytochemical analysis, it was determined that Moringa oleifera Lam. extracts contains

flavonoids, tannins and alkaloid which is a potential toothpaste component.

Recommendations

1. Future studies using other solvents for extraction may be conducted on same varieties of

malunggay.

2. Extraction method using mortar and pestle is recommended.

3. Based from the results of the phytochemical analysis, toothpaste with ground malunggay

may be developed.

References

American Academy of Periodontology. (2015). FlossYour Teeth - On the Double. Retrieved from

https://www.perio.org/consumer/flossing08

Caurez, C.L. (2012). Antibacterial potential of malunggay (moringa oleifera Lam.) root extracts.

Batangas State University (Philippines).

Chirumbolo, S. (2010). The Role of Quercetin, Flavonols and Flavones in Modulating

Inflammatory Cell Function. Inflammation & Allergy - Drug Targets IADT, 9(4), 263-285

Chirumbolo, S. (2011). Plant polyphenolic compounds as potential antimicrobial drugs. Journal

of Medical Microbiology, 60(10), 1562-1563

Cheok, C. Y., Karim salman, K. A., & Rabiha, S. (2014). Extraction and Quantification of Saponins: A Review. Department of Food Technology, Faculty of Food Science and

Technology, University Putra Malaysia

Chunli S., Zhengshuang W., Ziyan W., & Hongcheng Z. (2015) Effect of Ethanol/Solvents on

Phenolic Profiles and Antioxidant Properties of Beijing Propolis Extracts (Vol. 2015)

Esser, K., Kandereit, J. W., Lüttge, U., & Runge, M. (2013). Progress in Botany: Genetics Cell

Biology and Physiology Systematics and Comparative Morphology Ecology and Vegetation

Science (Vol. 60). Springer Verlag.

Fox, M. (2015). Sterol Functions. Retrieved May 14, 2016, from

http://www.livestrong.com/article/341576-sterol-functions/

Fuglie, L. J. (2000,). New Uses of Moringa Studied in Nicaragua. ECHO Development Notes #68.

Retrieved from

http://www.echotech.org/network/modules.php/name=New&file=article&sid=194

Simon. L, (2007). The Role of Streptococcus Mutans and Oral Ecology in the Formation of

Dental Caries.Yale University

Main, L. (2015). Stanols and Sterols (N. Ozerianskaya, L. Rout, & A. Atrey, Eds.). British

Dietetic Association. Retrieved from

https://www.bda.uk.com/foodfacts/PlantStanolsAndSterols.pdf

Malunggay. (2013). Retrieved from

http://cagayandeoro.da.gov.ph/wp-content/uploads/2013/04/MALUNGGAY.pdf

Marero, L. M. (2014). A Second Look at the Lowly Malunggay. Food and Nutrition Research

Institute

Margaret, R. F. (2013). Alkaloids: Biochemistry, Ecology, and Medicinal Apllication. Springer

Science and Business Media

Medical Health Guide. (2016). Malunggay. Retrieved from

www.medicalhealthguide.com/articles/malunggay.htm

Nutriology your health to the molecular level. Dental Health Foundation. Retrieved from

http://www.nutriology.com/glycoside.html

Özçelik, B., Aslan, S., Kartal, M., Karaoglu, T., Şener, B, & Choudhary, M. I. (2009). In vitro

biological activity screening of Lycopodium complanatum L. ssp. chamaecyparissus (A. Br.)

Döll. Natural Product Research, 23(6), 514-526.

Pandey S., & Chadna, A. (1993). A Textbook of Botany: Plant Anatomy and Economic Botany, 3,

538. Vikas Publishing House Pvt Ltd.

Pharmar S., Sharma T., Airo V., Bhatt R., Aghara R., Chavda S., & Gangwal A.

(2013). Neuropharmacological Effects of Triterpenoids, 4(2), 354-372.

Pietta, G. P. (2000). Flavonoids as Antioxidant, 63(7), 1035-1042.

Rhaman, M. M., Sheikh, M. I., Sharmin, S. A., Islam, M. S., Rahman, M. A., Rahman, M. M., &

Alam, M. F. (2009). Antibacterial Activity of Leaf Juice and Extracts of Moringa oleifera

Lam. against Some Human Pathogenic Bacteria.CMU J Nat Sci, 8(2), 219.

Sheiksh M. R., I., Sharmin, S. A., Islam, S. M., Rahman, A. M., & M. M. (2009). Antibacterial

Activity of Leaf Juice and Extracts of Moringa oleifera Lam. Against Some Human

Pathogenic Bacteria. 8(2), 219.

Shachi, S., & Verma, S. S. (2011). Antibacterial Properties of Alkaloid Rich Fractions Obtained

from Various Parts of Prosopis Juliflora, 2(3), 114-120.

Kakhia, T. (2012). Alkaloid and Alkaloid Plants. Adana University-Industry Joint Research

Center

O'maille, P., Thimmappa, R., Geisler, K., Louveau, T & Osbourn, A. (2014). Triterpene

Biosynthesis in Plants. Annual Review of Plant Biology Annu. Rev. Plant Biol., 65(1),

225-257. doi:10.1146/annurev-arplant-050312-120229

Tokajuk, D. (2006). Aerobic and Anaerobic Bacteria in Subgingival and Supragingival Plaques of

Adult Patients with Periodontal Disease,51(1).

Tomsone, L., Kruma, Z., & Galoburda, R. (2012). Comparison of Different Solvents and

Extraction Methods for Isolation of Phenolic Compounds from Horseradish Roots

(Armoracia Rusticana), 6(4).

Vignais, P., & Vignais, P. M. (2010). Discovering life, manufacturing life: How the experimental

method shaped life sciences. Dordrecht: Springer.

Villafuerte, N. F. (2015). Malunggay (moringa)-miracle plant (1). Manila Bulletin

Woudstra, W. J. (2015, October 27). Oral Bacteria: What Lives in Your Mouth?. Colgate Care

Center. Retrieved from

http://www.colgate.com/en/us/oc/oral-health/basics/mouth-and-teeth-anatomy/article/oral-bac

teria-what-lives-in-your-mouth-0513

WikiPiipinas Hip ‘nfree Philippine encyclopedia. (2010). Retrieved from

Tagalog.wikipilipinas.org/index.php/Malunggay

YGOY Health Community. (2015). The 5 Stages of Digestion. Retrieved from

http://digestion.ygoy.com/2010/07/15/the-5-stages-of-digestion/

Taclan, L B., Balila J. S., Balagtas M. Aclan, E. M., Orbon, M. C. Taclan, E. Y., Centeno, I. A.

(2016). Drying Kinetics o Okara (Soy Pulp) Using the Multi-Commodity Heat Pump Dryer

(MCHPD). Word Academmy o Science, Engineering and Technology.