RESEARCH TOPIC PROPOSAL

Students Name: Justin Albert A. Garcia

Date of Submission: September 22, 2017

Program: HUB41

Course Code and Title: ENGL102a Information Literacy

Research Topic: Effect of Gumamela Extract to Kidney Damage

Primary Research Objective: To determine the effect of crude flower extract from Hibiscus rosa-

sinensis L. (Gumamela) in acetylsalicylic acid-induced kidney damage in Rattus norvegicus

(Albino Rat)

Research Title: Effect of Crude Flower Extract from Hibiscus rosa-sinensis L. (Gumamela) In

Acetylsalicylic Acid-Induced Kidney Damage in Rattus norvegicus (Albino Rat)

Specific Research Questions

1. To determine the protective potentials of crude flower extract from Gumamela on aspirin-

induced kidney damage in terms of Serum Creatinine.

2. To determine the curative potentials of crude flower extract from Gumamela on aspirin-

induced kidney damage in terms of Serum Creatinine.

References:

Ali BH, Cahlikova L, Opletal L, Karaca T, Suleimani YA, Al Zaabi MH, Nemmar A, Blunden G
(2017). Effect of Aqueous Extract and Anthocyanins of Calyces of Hibiscus sabdariffa L.
(Malvaceae) in Rats with Adenine Induced Chronic Kidney Disease. The FASEB
Journal. 31, 1. http://www.fasebj.org/content/31/1_Supplement/819.7.abstract
Ademiluyi AO, Oboh G, Agbebi O, Akinyemi AJ. 2013. Anthocyanin Rich Red Dye of Hibiscus
sabdariffa calyx modulates cisplatin-induced nephrotoxicity and oxidative Stress in rats.
International Journal of Biomedical science. 9(4):243-248.
Altintas R, Polat A, Parlakpinar H, Vardi N, Beytur A, Oguz F, Sagir M, Yildiz A, Duran Z. 2013.
The effect of melatonin on acetylsalicylic acid-induced kidney and testis damage. Human
& Experimental Toxicology. 33(4):383395.
Arullappan S, Zakaria Z, Basri DF. 2009. Preliminary screening of antibacterial activity using
crude extracts of Hibiscus rosa-sinensis. Tropical Life Sciences Research. 20(2):109-118.
Asis CV. 1996. Plants of the philippines. Manila: Pundasyon sa Pagpapaunlad ng Kaalaman sa
Pagtuturo ng Agham. Page 268.
Austria JL, Manalo A. 2005. Effectiveness of crude anthocyanin extract of Hibiscus rosa-sinensis
in the protection of acetaminophen-induced liver damage in albino mice [dissertation]. De
La Salle University-Dasmarias.
Brickell C. 2008. RHS A-Z encyclopedia of garden plants. London:Kindersley. Page 1136.
Hou Z, Qin P, Ren G. 2010. Effect of anthocyanin-rich extract from black rice (Oryza sativa l.
japonica) on chronically alcohol-induced liver damage in rats. J. Agric. Food Chem.
Journal of Agricultural and Food Chemistry. 58(5):31913196.
Yusoff A, Kumara NTRN, Lim A, Ekanayake P, Tennakoon KU. 2014. Impacts of temperature
on the stability of tropical plant pigments as sensitizers for dye sensitized solar cells.
Journal of Biophysics. 2014:1-8.
Ibrahim UK, Muhammad II, Salleh RM. 2011. The effect of ph on color behavior of brassica
oleracea anthocyanin. Journal of Applied Sciences, 11: 2406-2410.
 METHODOLOGY

3.1 Research Design

This study was entirely experimental in nature by which the method of data collection was
Randomized Complete Block Design (RCBD). It aimed to determine the effectiveness of crude
flower extract from Hibiscus rosa-sinensis against acetylsalicylic acid-induced kidney damage in
albino rat. The serum study was carried out within a day of experimentation.
The RCBD consisted of six blocks. Each block consisted of one rat from each test group,
from T0 to T3. Each block was tested for its Serum Creatinine (Cr), and the mean of the results
and standard deviation.

3.2 Research Procedure

Preparation of Test Animals

Acquisition of Materials
Twenty 6-to-8 weeks old, healthy male albino rats was purchased from the Animal Clinic
at Market Place Ave., Dasmarias, Cavite. The drug that was used to induce damage,
Acetylsalicylic acid (Aspirin), was purchased from the commercial drugstore in Dasmarias,
Cavite. The plant where anthocyanin was extracted, Hibiscus rosa-sinensis (Gumamela) petals,
approximately 1 kg, was used.
Acclimatization of Albino Rats
The researchers allowed the albino rats to adjust in the new environment after acquiring
twenty-four 6-to-8 weeks old test specimen. Wire mesh was used to make the cages where the test
specimen was housed, with a diameter of 6x6x6 inches. The animals were allowed to acclimatize
for seven days. Throughout the acclimatization period, test animals were given 5 g of pellets each.
They were fed every 6 hours (three times a day) specifically at 6 am, 12nn, and 6 pm. Water was
also given to the test animals at the same time as the pellets. To make sure that the place will be
contamination-free, leftovers and animal feces were removed from time to time.

Extraction of Anthocyanin from Hibiscus rosa-sinensis

Approximately 1 kg of Hibiscus rosa-sinensis (Gumamela) petals were collected. The
petals were cleaned, then immersed with 75% methanol for 2 days. Crude extract from the petals
passed through cheesecloth filtration three times. The filtered extract was placed in the rotary
evaporator for further extraction (Austria and Manalo 2005). The liquid filtrate was used as the
crude flower extract.

Determination of the Presence of Anthocyanin from Crude Extract

After undergoing the rotary evaporator, the collected crude flower extract was subjected to
a test, which distinguished anthocyanin from betacyanin. The first test is to heat it with 2M of HCl
for at least 5 minutes at 100oC. Crude flower extract should have a stable dark red color. Another
test that was conducted was to add 2M of NaOH, dropwise, crude flower extract should change
from blue to green and slowly fade (Austria and Manalo 2005).
Preparation and Administration of Treatment
The tablet was pounded using mortar and pestle. It was weighed using the analytical
balance depending on the amount needed (200mg/kg). Gavage feeding was done to treat the test
animals with 200mg/kg aspirin and 100mL/kg crude flower extract, which involved the insertion
of a flexible ball tip needle into the mouth of the rat. Once the rat is properly restrained, the gavage
needle was carefully inserted in the mouth and dosage was delivered (Austria and Manalo 2005).

Multiple Dose Treatment

The next day after the last day of acclimatization, a test was done to determine the
protective and curative effect of crude flower extract to the damage done by high dosage of
acetylsalicylic acid to the kidney of the albino rat. Test animals was divided into four test groups
of five animals in each cage. Blood was extracted via tail section from each rat before all
treatments to get the baseline. The first group, T0, was given a single dose of 200mg/kg aspirin,
and was given normal saline solution for every 12 hours for 48 hours, 1 hour after the
administration of aspirin. The second group, T1, was given normal saline solution for every 12
hours for 48 hours, and was administered a single dose of 200mg/kg of acetylsalicylic acid, 1 hour
after the last dosage of normal saline solution. The third group, T2, was administered 100 mg/kg
of crude flower extract for every 12 hours for 48 hours, 1 hour after the administration of 200mg/kg
acetylsalicylic acid. And the fourth group, T3, was administered 100 mg/kg of crude flower extract
for every 12 hours for 48 hours, 1 hour before the administration of 200mg/kg acetylsalicylic acid.
The doses of anthocyanin and acetylsalicylic acid was determined through the previous studies
(Austria and Manalo 2005; Atlintas 2013).

Blood Sample Extraction

Four hours after the multiple dose treatment, blood sample was taken from the tail section
of the rat. The rat was placed in a jar filled with cotton soaked with 300L isoflurane. When the
rats breathing slowed down and was lying on its side, it was quickly taken out of the jar and was
put in a restraining tube that covered the forelimbs to the hind limbs. A scalpel will then be used
to cut at most 1 cm of the tail section. 2mL of blood sample will then be collected using capillary
tube. The area of extraction was applied with antiseptic and pressure afterwards to stop the
bleeding. The collected blood samples were placed in a cool insulated container.

3.3 Data Gathering

The collection and testing of blood was done with the assistance of the supervising vet med
before all treatments and after four hours of giving the last dosage of the respective treatment given
to each test groups. The amount of blood that was tested was 2mL as per the instruction of the vet
med. The blood was placed on a Dialab Autolyser to profile the blood to get the amount of Serum
Creatinine.

3.4 Statistical Analysis

Serum Creatinine (Cr) was the dependent variable that was measured. It was measured through
laboratory testing. Paired Two Sample for Means t-Test was used to determine whether the means
of two independent groups differ, and calculated a range of values that is likely to test the
hypotheses. The difference of the means of baseline and post-treatment Serum creatinine was
calculated by subtracting the baseline Serum creatinine to the post-treatment Cr. The difference
between the means of post-treatment and baseline of groups T0 and T2 were compared to determine
the curative effect of the crude flower extract, while the difference between the means of post-
treatment and baseline of groups of Serum creatinine in T1 and T3 were compared to determine the
preventive effect of the crude flower extract.

RELATED STUDIES

1. A study on the effects of Anthocyanin-Rich extract from Black Rice (Oryza sativa L. japonica)
on chronically alcohol-induced liver damage in rats showed that rats treated with AEBR
(anthocyanin-rich extract from black rice) showed a better profile of the antioxidant system.
The results also showed that the introduction of anthocyanin alongside ethanol had lower
hepatic markers compared to the alcohol-only group. It showed that AEBR has a beneficial
effect in the adverse effect of alcohol. The study shows that anthocyanin can be an antioxidant,
thus can may also be possibly used as a protective or curative measure for other organs of the
body like the kidney (Hou et al. 2010).

2. A study conducted as to which of the 35 species of plants containing anthocyanin pigments

could be used as a potential sensitizer for Dye Sensitized Solar Cell, Hibiscus rosa-sinensis
was found to have the second-highest absorption peak. It was found out that at 4oC,
significantly greater stability of extracted anthocyanin pigment was shown. Half-life periods
for anthocyanin also decreased when stored at a higher temperature. This study tells that there
is a presence of anthocyanin pigment in Hibiscus rosa-sinensis (Yusoff et al. 2014).

3. Anthocyanins are flavonoids that are natural colorants, they can be used as pH indicators
because their colors change depending upon the pH of their environment. Anthocyanin turns
red-pink in acidic solutions (pH 1-6), and turn green in alkaline or basic solutions (pH 8-14).
The increase of the pH values from 1 to 7 changes the color of the extract to a more purplish
tone, while the increase of pH values greater than 7 shows a change of color to green
(Ibrahim 2011).