Study in Activity Combination of Physalis angulata and Hibiscus

sabdariffa in 70% Ethanol Extract to Decrease Blood Sugar Levels

and Histopathology of Pancreas Langerhans Island in Alloxan

Induced Diabetic Rats

Hadi Sunaryo, Ni Putu Ermi Hikmawanti, Hesty Awanis Listyaningrum

Faculty of Pharmacy and Science, Universitas Muhammadiyah Prof. Dr. Hamka

Keywords: Physalis angulata L; Hibiscus sabdariffa L; Blood sugar; Pancreatic Islet of Langerhans

Abstract: Ciplukan (Physalis angulata L) and roselle petals (Hibiscus sabdariffa L) have been shown to have blood

sugar levels decreasing properties. This study aims to determine the effect of the Ciplukan and Roselle

petals ethanol extract combination to the decrease in blood sugar levels in alloxan-induced diabetic rats

(Sprague Dawley). The diabetic rats were divided into seven groups, each consisting of four rats. Group I

(negative control), group II (positive control), group III (ciplukan 10mg/200g BW), group IV (roselle petals

60mg/200g BW), group V (ciplukan 5mg/200g BW and roselle petals 30mg/200g BW), group VI (ciplukan

5mg/200g BW and roselle petals 60mg/200g BW) and group VII (ciplukan 10mg/200g BW and rosella

petals 30mg/200g BW). All groups were induced with alloxan. Parameters observed were decreased blood

sugar levels on the 15th and 30th day. After that, surgery and histopathological observation of pancreatic

organs were conducted at the 31st day. Blood sugar percentage was analysed using one-way ANOVA with

95% significance level and continued with the Tukey test. The results showed that group number VII

combination of 70% ethanol extract ciplukan with 10mg/200g BW dose and roselle flower petal dose

30mg/200 g BW decreased blood sugar level equal to glibenclamide dose 0.052mg/200g BW (p≥0.05) that

is equal to 66.74% and improvement activity of pancreatic islets of Langerhans, has been shown at the

histology result.

1 INTRODUCTION

Diabetes mellitus (DM) is a disease or disorder of

fat, carbohydrate, and protein metabolism caused by

a disturbance of insulin secretion, insulin work

(sensitivity), or both characterized by

hyperglycemia. Symptoms in DM patients such as

polyuria, polydipsia, polyphagia, weight loss, and

lethargy by hyperglycemia are the most common

early symptoms (Dipiro et al., 2014).

Synthetic antidiabetic drugs have side effects of

gastrointestinal disorders, anorexia, lactic acidosis,

vitamin B12 absorption disorders, and edema. Plants

as traditional medicine are easy to obtain and

generally has lower side effects when compared to

synthetic drugs. Components in one ingredient

(usually) have mutually supportive effects, and one

plant has multiple pharmacological effects that

makes it more suitable and safer for degenerative

metabolic disease (MOH RI, 2008). The ciplukan

(Physalis angulata L.) and roselle petals (Hibiscus

sabdariffa L.) are alternatives that can be used in the

treatment of DM (Sunaryo et al., 2012; Rosemary et

al., 2014).

Sunaryo et al. (2012) reported that the fraction of

chloroform ciplukan with the dose of 2mg/20g BW

in mice has the antidiabetic effect and can improve

the number of Langerhans pancreatic cells

proportional to glibenclamide. Rosemary et al.

(2014) reported that ethanol extract of roselle petals

at 600mg/kg BW dose was able to lower blood sugar

levels comparable with glibenclamide of 77.69% in

streptozocin-induced mice.

Based on this background, this study wanted to

prove the effect of ciplukan and roselle petals

ethanol extract in combination to the lowering of

sugar levels in alloxan-induced rats. The purpose of

combining the two extracts is to obtain smaller doses

and have activities that are comparable or even

greater than the single herbal extract.

Study in Activity Combination of Physalis angulata and Hibiscus sabdariffa in 70 .

DOI: 10.5220/0008240401170122

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 117-122

ISBN: 978-989-758-349-0

117

2 MATERIALS AND METHODS

2.1 Plant and Preparation of Extracts

Physalis angulata L. was obtained from Merapi

Farma Herbal Yogyakarta, and Hibiscus sabdariffa

L. obtained from Balai Tanaman Obat dan Rempah

(BALITTRO) Bogor. The reference drug used was

glibenclamide. A total of 1500 g of dried herb

ciplukan and 1500 g of dried roselle petals each

made into a powder. It was then sieved with mesh

no. 40, weighed, and recorded. As many as 900 g

and 1000 g of powder were each macerated by using

ethanol 70%. They were left soaking for the first 6

hours while stirring occasionally, and then let stand

for 18 hours. The maceration was performed three

repetitions using a new liquid. The macerate was

then evaporated using a vacuum rotary evaporator

until it turns into a viscous extract (MOH RI, 2008).

2.2 Phytochemical Screening

Phytochemical screening was performed using TLC

method by preparing a 10 mg/ml test solution in

ethanol for each extract. Prepared test solutions were

bottled on a silica gel TLC plate GF254. The

separation and detection systems of the compounds

are shown in Table 1.

2.3 Preparation of Animal

The animals used for study were white male rats of

Sprague Dawley strain, aged 2-3 months with a body

weight around 150-300 g, obtained from the Bogor

Agricultural University (IPB) of the Production

Division of Working Meat and Animal Livestock.

The experiment was completed with a randomized

design, using 28 male rats divided into seven groups

consisting of four rats. Before the experimental

treatment, the animals were first acclimatized in the

cage with standard feed and drink for approximately

seven days. The protocol no. 17-05-0486 has been

approved by the Health Research Ethics Committee

of the Faculty of Medicine, Universitas Indonesia

no. 453/UN2.F1/ETIK/2017.

2.4 Measurement of Blood Sugar

Levels

The rats fasted for 12 hours before treatment. Blood

sample was collected through the eye area (sinus

orbital). Blood glucose measurements were

performed on day 15 and after administration of the

test substance on day 30 using a spectrophotometer

clinical 300.

2.5 Observation of Pancreatic

Histopathology

The pancreatic histopathological observation process

began with surgery in rats that have been

anaesthetized with ketamine. The preparation stage

of preparation included the taking of pancreatic

organs, fixation, dehydration, clearing, embedding,

cutting, staining with Haematoxylin-Eosin, closing

the dosage, and observation by microscope

(Olympus x21) with 4x10 magnification.

2.6 Data analysis

The percentage data of decreased blood sugar levels

was statistically tested using ANOVA one way with

Table 1. Separation & detection system with TLC method

Compounds Mobile Phase Detection Reagent

Alkaloid Ethyl acetate:methanol:ammonia

(4:4:1)

Dragendroff

Saponin Chloroform:methanol

(1: 4)

Vanillin-Sulfuric acid

Tanin Ethyl acetate: methanol :

Formic acid 5%

(

4: 1: 1

)

FeCl

Flavonoid Acetic Acid: methanol :

formic acid 5%

(

4: 1: 1

)

Cytro-borate 5%

Terpenoid n-hexane: Ethyl acetate

(4: 1)

Liebermann-Bouchard

118

95% significance level. Then proceeded with the

Tukey test to determine the significant differences

between groups.

3 RESULTS AND DISCUSSION

3.1 Physalis angulata L. and Hibiscus

sabdariffa L. extracts

Table 2 shows the results of the extract of ciplukan

herb and roselle petals. Preparation of the dried

powder was carried to enlarge its surface area so that

the solvent used can be readily absorbed into dried

powder and the active compound which is attracted

more optimally (Vitasari, 2012), while pollination

will help break the walls and cell membranes to

maximize the extraction process (Koirewoa et al.,

2016).

The dried powder was extracted by maceration

using 70% ethanol. Ethanol is considered to be a

more selective (mouldy and bacteria growing in

ethanol 20% and above), non-toxic, neutral, and

well-absorbed (Pratiwi, 2010). Using ethanol as a

universal solvent is due to its easy-to-dissolve of

polar, semi-polar, and non-polar active substances

and its ability to precipitate proteins and inhibit the

action of enzymes to avoid hydrolysis and oxidation

processes (Dharma et al., 2010). According to

Indraswari (2008), ethanol with 70% concentration

is effective in attracting active compounds. The

ethanol solvent has two sides consisting of a polar

OH-group and a non-polar CH

group. The

extraction by maceration was done because this

method can be used for large quantities of samples,

the implementation is simple, does not require

special treatment and the possibility of

decomposition of the active substance by the

influence of temperature can be avoided because

there is no heating process (Dharma et al., 2010).

Percentage of extract weight to dried powder shows

the effectiveness of the extraction process. The

effectiveness of the extraction process is influenced

by the type of solvent, particle size of dried leaves

powder, method and duration of extraction

(Istiqomah, 2013). The longer the extraction time,

the higher the saturation point of the solution. The

contact between the sample and the solvent can be

increased while it was shaking, to keep the contact

between the sample and the solvent frequent,

resulting in a more perfect extraction process

(Koirewoa et al., 2016). High percentage of extract

weight to dried powder results indicate the

possibility of the chemical compounds contained in

the extract is also high (Isnawati et al., 2006)

3.2 Phytochemical Screening

Phytochemical screening in this study used the thin

layer chromatography (TLC) method. The purpose

of using TLC is for more accurate and better

identification of compounds contained in the extract.

TLC is a way of physical separation with elements

to be separated distributed between two phases

(stationary phase and mobile phase). Separation is

based on migration differences and distribution of

compounds or ions (Astuti, 2016). The silica gel

plate GF254 was used as stationary phase, while the

eluent was used as the mobile phase varies for each

of the secondary metabolites to be tested. A good

eluent can separate the compound in large quantities

that are marked by the appearance of stains. The

stain that formed was not tailed, and the distance

between the stains with each other was clear

(Harborne, 1987). TLC plates were activated by the

oven at temperature 115°C for 15 minutes to remove

water contained in KLT plate (Sastrohamidjojo,

2007).

The results of screening tests can be found at

Table 3. Stains obtained from the TLC were still not

Table 2. Results of Ciplukan Herb Extraction and Rosella

Flower Petals

No Name Physalis

angulata L.

herb extract

Hibiscus

sabdariffa L.

flower petals

extract

1 Extract

weight

98.80 g 234 g

Percentage

of extract

weight to

dried

owde

10.98 % 23.4 %

Table 3. Phytochemical Screening Results

No. Compounds Physalis

angulata L.

Hibiscus

sabdariffa L.

1 Alkaloids + -

2 Flavonoids + +

3 Saponins - -

4 Tannin + -

5 Terpenoids + +

Note: (+) = detected

(-) = not detected

Study in Activity Combination of Physalis angulata and Hibiscus sabdariffa in 70

119

completely separated because the metabolite

compounds were not yet perfectly attracted, possibly

due to unsuitable eluents, inappropriate selection of

TLC plates, and extract concentrations that were too

high.

3.3 Results Measurement of Blood

Sugar Levels

The result of blood glucose levels of each group

before and after treatment can be seen in Figure 1.

The percentage data of decreased blood sugar levels

(Table 4) were inserted into statistics to test and

determine their normality and homogeneity with

Kolmogorov and Smirnov. The data of group I is not

included in statistics because the results were

different.

The data analysis showed normal data

distribution {(p=0.819) ≥0.05} and homogeneity

{(p=0.418) ≥0.05}. The analysis was continued by

ANOVA one-way test to find out whether there was

any significant difference or not in each treatment

group with p≤0.05. Based on the analysis results, the

value obtained was {(p=0.044) ≤0.05}. These results

indicate that giving a combination of herbal extract

ciplukan and roselle petals have a significant effect

on the decrease in blood sugar levels of diabetic rats.

The data then continued with the Tukey test to

know the difference between each treatment groups.

Based on the Tukey test, there was no significant

difference (p≥0.05) between the positive control

group and single ciplukan group, single roselle petal

extract, combination I, combination II and

combination III. This result means that in this case,

all test groups have activities that are proportional to

Table 4. Average percentage of reduced blood glucose

levels

Treatment groups Reduced blood glucose

levels

(

)

± SD

ative 2.86 ± 0.81

Positive 63.02 ± 2.83

Ciplukan ext

act 60.86 ± 3.65

Roselle petals extract 60.12 ± 0.02

Combination I 56.77 ± 5.99

Combination II 60.50 ± 3.80

Combination III 66.74 ± 4.02

Note: The test was performed with four repetitions.

Figure 1. Graph of average blood glucose levels (mg/dl)

of each group before and after treatment. (I) Negative

control group. (II) Glibenclamid dose 0.052mg/200g BW

group. (III) Extract Ciplukan dose 10mg/200g BW

group.(IV) Extract Roselle petals dose 60mg/200g BW

group. (V) Combination I (½ dose of Ciplukan: ½ dose of

Roselle petals) group. (VI) Combination II (½ dose of

Ciplukan: 1 dose of Roselle petals) group. (VII)

Combination III (1 dose of Ciplukan: ½ dose of Roselle

petals) group.

Figure 2. Histology of Pancreatic Islets Diabetic Rats

with Magnification 4x10: (A) The arrows indicate the

empty spaces (necrosis) of the Langerhans pancreas

island cell in the negative control group; (B) Positive

control group, no necrosis; (C) Ciplukan Extract

group; (D) Flower Rosela extract group; (E)

Combination I group; (F) Combination II group; (G)

Combination III group

120

the positive control of glibenclamide in lowering

blood sugar levels.

3.4 Observation of Pancreatic

Histopathology

The results of pancreatic histologic preparations can

be seen in Figure 2. Necrosis is cell or tissue death

due to a reversible degeneration process. This

condition can be caused by several things including

toxins, drugs, low blood supply, no nerve

preservation, temperature, light, radioactive and

mechanical trauma (Berata et al., 2011). The alloxan

mechanism in principle occurs through several

processes that stimulant produce damage effects on

pancreatic β cells. The reactive oxygen that is

formed can cause damage to pancreatic beta cells.

The destruction of pancreatic beta cells made a

decreased insulin secretion (Szukudelski, 2001).

The results of this study have shown that the

decrease in blood sugar levels and improvement of

the pancreatic Langerhans islet cells induced the

insulin secretion to compensate for high blood

glucose levels and subsequently lowered it. The

mechanism of decreased blood sugar levels and the

improvement of the islet cells of Langerhans were

derived from the compounds of flavonoids,

anthocyanins, and terpenoids contained in ethanol

extract 70% of ciplukan and roselle petals.

Flavonoids are antioxidants that can clean up

excessive free radicals, break the chain of free

radical reactions, and binding metal ions (chelating).

Flavonoids also have an inhibitory effect on the α-

glucosidase enzyme (Taufiqurrohman, 2015).

Anthocyanin has hypoglycaemic activity by

inhibiting α-glucosidase in the intestinal lumen and

improving insulin sensitivity (Kowalczyk et al.,

2013). Terpenoids have antidiabetic activity that can

stimulate the regeneration of Langerhans island cells

so that the cell damage of Langerhans Island in

particular β cells can be reduced gradually, the

increased number of Langerhans island cells also

allows the secretion of insulin so as to compensate

for high blood sugar levels and then decrease it

(Sunaryo et al., 2012).

4 CONCLUSIONS

The combination of 70% ethanol extract of ciplukan

and roselle petals successfully decreased blood sugar

levels in alloxan-induced rats. The combination of

70% ethanol extract of ciplukan and roselle flower

petals in dose I showed lower blood sugar level,

combination dose II equal to 60.50%, and

combination dose III equal to 66.74%.

Based on the statistical study, there was no

significant difference (p≥0.05) between the positive

control group and single ciplukan group, single

roselle petal, combination I, combination II and

combination III. This result means that in this case

all test groups have activities that are proportional to

the positive control of glibenclamide in lowering

blood sugar levels.

The combination of 70% ethanol extract of

ciplukan and roselle petals was able to provide an

improvement of the Langerhans island cell in

alloxan-induced rats compared with the negative

control group.

REFERENCES

Astuti, S. 2016. Uji Aktivitas Antioksidan Ekstrak

Kloroform Daun Tomat (Solanum lycopersicum L.),

Daun Cabai Merah (Capsicum annum L.) dan Daun

Ciplukan (Physalis angulata L.) Dengan Metode

DPPH. Tugas Akhir. FMIPA Universitas Sebelas

Maret. Surakarta

Berata IK, Winaya IBO, Adi AAAM, Adnyana IBW.

2011. Patologi Veteriner Umum. Denpasar: Swata

Nulus

Departemen Kesehatan RI. 2008. Tingkat Manfaat

Keamanan dan Efektifitas Tanaman Obat dan Obat

Tradisional. Tawangmangu: Departemen Kesehatan

RI. Hlm. 5

Dharma S, Erdinal, Rizal Z. 2010. Pengaruh Pemberian

Ekstrak Etanol Kelopak Bunga Rosella (Hibiscus

sabdariffa L.) Terhadap Kadar LDL pada Mencit

Putih Betina. Jurnal Farmasi Higea. Vol. 2, No.2.

Universitas Andalas. Padang

Dipiro JT, Talbert RL, Yee GC, Matzke GR. 2014.

Pharmacotherapy: A Pathophysiologic Approach.

New York: The McGraw-Hill Companies. Hlm. 2547-

2548

Harborne JB. 1987. Metode Fitokimia Penuntun Cara

Modern Menganalisa Tumbuhan. Jilid II. ITB.

Bandung

Isnawati A, Raini M, Alegantina S. 2006. Standarisasi

Simplisia Ekstrak Etanol Daun Sembung (Blumea

balsamifera L.) Dari Tiga Tempat Tumbuh. Media

Litbang Kesehatan XVI, No.2

Istiqomah. 2013. Perbandingan Metode Ekstraksi

Maserasi dan Sokletasi Terhadap Kadar Piperin Buah

Cabe Jawa (Piperis retrofracti fructus). Skripsi. FKIK

UIN Syarif Hidayatullah. Jakarta

Kowalczyk E, Krzesinski P, Kura M, Szmigiel B,

Blaszczyk J. 2003. Anthocyanins in Medicine. J

Pharmacol. (55): 699-702

Pratiwi, E. 2010. Perbandingan Metode Maserasi,

Remaserasi, Perkolasi dan Reperkolasi Dalam

Ekstraksi Senyawa Aktif Andrographolide Dari

Study in Activity Combination of Physalis angulata and Hibiscus sabdariffa in 70

121

Tanaman Sambiloto (Andrographis paniculata

(Burm.f.) Nees). Skripsi. Fakultas Teknologi Pertanian

IPB, Bogor. Hlm. 8-9

Rosemary, Rosidah, Haro G. 2014. Antidiabetic Effect of

Rosella Calyces Extract (Hibiscus

sabdariffa L.) in Streptozocin Induced Mice.

International Journal of PharmTechResearch. Coden

(USA)

Sastrohamidjojo, H. 2007. Dasar-Dasar Spektrosfotokopi,

Edisi kedua, Cetakan kedua. Yogyakarta: Penerbit

Liberty

Sunaryo H, Kusmardi, Trianingsih W. 2012. Uji Aktivitas

Antidiabetes Senyawa Aktif Dari Fraksi Kloroform

Herba Ciplukan (Physalis angulata L.) Terhadap

Penurunan Kadar Glukosa Darah dan Perbaikan Sel

Langerhans Pankreas pada Mencit yang Diinduksi

Aloksan. Jakarta: Farmasains. 1 (5). Hlm. 246-251

Szkuldelski, T. 2001. The Mechanism of Alloxan and

Streptozotocin Action in β-cells of The Rat Pancreas.

Physiol. Res. 50: 536-546

Taufiqurrohman. 2015. Indonesian Bay Leaves As

Antidiabetic For Type 2 Diebetes Mellitus. Article

review. Vol. 4, No.3. Fakultas Kedokteran Lampung

Vitasari ON. 2012. Uji Aktivitas Antibakteri Ekstrak

Etanol Daun Ceplukan (Physalis angulata L.)

Terhadap Staphylococcus aureus dan Pseudomonas

aeruginosa. Tugas Akhir. FMIPA Universitas Sebelas

Maret, Surakarta

122