1. Aside from this, ancient herbal remedies are

 

1. Introduction

            Plants
are endowed with a potential to produce a wide range of allele-chemicals or
also known as phytochemicals, which has significant and valuable therapeutic
potential. It is because these phytochemicals cab exhibit antioxidant,
antimicrobial, anti-inflammatory, etc. In recent years, humans have incredibly
witnessed a rapid production of new and/or enhanced reports and information on
plants and their natural products with regards to their therapeutic potential. Incidentally,
researchers around the world are engaged in a mission to hunt for novel
phytochemicals that could potentially be utilized in the management and
treatment of common diseases and illnesses in humans. Aside from this, ancient
herbal remedies are being re-evaluated and screened more efficiently to be
considered as potent and better alternative for over-dependence on conventional
and chemical-based treatments and remedies which often leads to antibiotic
resistance of pathogenic and dangerous microorganisms and too much dependence
worsens the condition of a human body by catastrophically interfering and
affecting with human biological activities.

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            The plant Cordyline fructicosa (L.) A. Cheval. belongs to the Family
Agavaceae. It is an erect, smooth shrub that may grow to a height of
approximately 3 m. The stems are generally non-branching and marked with
prominent leaf scars. The leaves are usually tinged red or purple, and are
crowded towards the upper part of the stem. The flowers are arranged in
clusters of 3 to 8 and they are pale stram colored and sweet-scented. It is
wildly cultivated throughout the Philippines for ornamental purposes.
Traditionally, it is used to treat diseases such as Hemoptysis due to pulmonary
tuberculosis, excessive menstrual discharge, hematuria, bleeding piles and
enteritis and bacillary dysentery. However, availability of enhanced research
to back up its therapeutic potential are scant.

            Based on this, the present study
aims to a) evaluate the antioxidant activity of the different fractions of
Cordyline fruticosa L. crude leaf
extract by the rate of scavenging the
DPPH radical and determination of total phenolics, and b) determine the
antibacterial property of the different fractions of C. fruticosa L. crude leaf extract against Staphylococcus aureus,
Bacillus subtilis, Streptococcus pneumoniae
and Klebsiella pneumoniae by well
diffusion method.

            The present study may add importance
to C. fruticosa L. itself which often
goes unnoticed which would naturally persuade people to preserve and to further
utilize the said species. The present study could also serve as scientific
basis to further the therapeutic importance of the C. fruticosa L.

2. Materials and Method

2.1. Sample Collection

            Cordyline
fruticosa leaves samples will be collected at the Pitogo
Elementary School and Pitogo High School, Pitogo, Makati City. The
collected samples will be washed thoroughly in tap water, cut into small
pieces, air dried at room temperature for 24 hours. Thereafter, it will also be
powdered using an electric blender and stored in sanitized plastic containers.
The sample will be then brought to the University of Philippines, Pedro Gil,
Manila for extraction and fractionation purposes. Taxonomic identification of
the plant sample will be authenticated by the Botany Division of the National
Museum of the Philippines.

2.2. Extraction and Fractionation of
the Leaf Sample

            The leaves will be oven- dried at 55
°C for 24 hrs. The oven- dried leaves will then be subjected to grounding and
ethanolic and methanolic crude extraction for 24 h. Then, filtration will be
subsequently done to obtain the ethanol and methanol crude extract which will
be then evaporated in a rotary evaporator set at 40 °C. The weight of the
residue will be noted and it will be dissolved with 100 ml distilled water. One-tenth
(10 mL) of the extract will be taken to serve as the crude ethanol and methanol
extract. Evaporation will performed and the weight of the residue will be noted
and then the concentration will be adjusted to 10, 000 ppm using methanol and
ethanol.

            The remaining 90 mL of the ethanolic
and methanolic crude extract will be subjected to solvent-solvent extraction
using an equal volume of n- hexane and ethyl acetate. Sodium sulfate will be
added in the n- hexane and ethyl acetate layers and allowed to stand overnight
to remove the remaining water. Thereafter, filtration will be performed to
remove the sodium sulfate. Afterwards, the filtrates will be evaporated in a
rotary evaporator set at 40 °C. The concentration of the extracts will be
adjusted to 10, 000 ppm using ethanol and methanol respectively.

2.3. Antioxidant Assay

2.3.1. DPPH Radical Scavenging Assay

            The antioxidant activity of the
plant extract will be estimated on the basis of the rate of scavenging activity
of the stable 1, 1- diphenyl 2-picrylhyorazyl (DPPH) free radical according to
the method described by Brand-Williams et al with slight modifications. 1ml
of  0.1 mM DPPH solution in methanol will
be mixed with 1ml of plant extract solution of varying concentrations ( 50,
100, 150, 200 and 250 µg/ml). Corresponding blank sample will be prepared and
L-Ascorbic acid (1-100 µg/ml) will be used as reference standard. Mixer of 1ml
methanol and 1ml DPPH solution will be used as control. The reaction will be
carried out in triplicate and the decrease in absorbance will be measured at
517nm after 30 minutes in dark using UV-Vis spectrophotometer. The inhibition %
will be calculated using the following formula.   

Inhibition
% = Ac-As/Ac×100

Where

Ac
is the absorbance of the control

As
is the absorbance of the sample

2.3.2. Determination of Total
Phenolics

            Using modified Folin-Ciocalteu
method, total phenol contents in the extract will be determined. An aliquot of
the extract will be mixed with 5 ml Folin-Ciocalteu reagent (previously diluted
with water at 1:10 v/v) and 4 ml (75 g/l) of sodium carbonate). The tubes will
be vortexed for 15 sec and be allowed to stand for 30 min at 40oC for color
development. Absorbance will be then measured at 765 nm using the Hewlett
Packard UV-VIS spectrophotometer. Samples of extracts will be evaluated at a
final concentration of 0.1 mg/ml. Total phenolic content will be expressed as
mg/g tannic acid equivalent using the following equation based on the
calibration curve: y = 0.1216x, R2 = 0.9365, where x was the absorbance and y
was the tannic acid equivalent (mg/g).

2.4. Antimicrobial Assay

2.4.1. Preparation
of Glassware and Isolation Room

            Glassware
apparatuses like test tubes, erlenmeyer flask, beaker, pipette, L-rod and petri
dish were sterilized in the autoclave for 15 minutes to assure that unwanted
organism were eliminated. The isolation room was disinfected using Zonrox and
wiped with 75 percent isopropyl alcohol. The researchers wore laboratory
outfits while inside the laboratory to ensure safety while performing and
handling media that have potent hazard.

2.4.2. Media
Preparation

Nutrient Agar

            A 22.5 g of nutrient agar (NA) were
dissolved in 500 ml of distilled water. It was sterilized in an autoclave at 15
psi for 15 minutes and then cooled at 45 °C before
pouring on plates.

2.4.3.
Plating
– Out Isolates

            Plating-out was done in a sterile
room pre-cleaned with UV lamp for 20 minutes with disinfectant such as alcohol
and chlorine-based materials. One milliliter (1 ml.) of each sample was dropped
on each plate using an L-shaped glass rod and incubated at 300C for
24 hours for the isolates to grow. Plating was used to spread the bacteria
present in the samples. Through this, overlapping of colonies was avoided.

2.4.4. Antibacterial Testing

            The n-hexane and ethyl acetate
fractions of both the ethanolic and methanolic crude extract will be tested
against Staphylococcus aureus, Bacillus
subtilis, Streptococcus pneumoniae and Klebsiella
pneumoniae for their antimicrobial activity through well diffusion method.

            The
prepared culture plates were inoculated with strains of the bacteria using
streak plate method. Wells were made on the agar surface with 10 mm cork borer.
The extracts were poured into the well using sterile syringe. The plates were
incubated at 35 °C for 24 hours. The plates
were observed for the zone clearance around the wells.

2.4.5. Reading
of Zones of Inhibition

            After 24 hours of incubation, the cleared
zone around the n-hexane and ethyl acetate fractions of both the ethanolic and
methanolic crude extract will be measured and the diameter of zone of
inhibition will be noted as well (mm) using Digital
Vernier Caliper. The data and results will be tabulated and analyzed. The
antimicrobial index (AI) will be computed using the following formula:

AI=

2.5.
Statistical Analysis

All
experimental will be run in triplicate, and statistical analysis will be
performed using SPSS software. Analysis of variance was performed by ANOVA
procedures. Results were given as means ± standard deviation. Significance
level was defined as P