RINGKASAN

OPTIMASI FORMULA SEDIAAN SIRUP MUKOLITIK IN

VITRO FRAKSI TERSTANDAR BUNGA KEMBANG SEPATU
(Hibiscus rosa-sinensis L.)
DISERTASI
Untuk memenuhi sebagian persyaratan
mencapai derajad Doktor (Dr.) pada program Doktor

Diajukan Oleh:
MIMIEK MURRUKMIHADI
08/276481/SFA/00032

Kepada
PROGRAM PASCASARJANA
PROGRAM STUDI ILMU FARMASI
FAKULTAS FARMASI
UNIVERSITAS GADJAH MADA
YOGYAKARTA
2012

ABSTRACT
Hibiscus Flower (Hibiscus rosa-sinensis L.) has been used traditionally
against cough. It was potential as secretolytic agent, but a standardized syrup is
still needed in order to obtain optimum pharmalogycal effect. The objective of
this research was to obtain an optimum standardized fraction syrup formula of
Hibiscus flower by Simplex Lattice Design Method, as well as to determine its in
vitro mucolytic activity and stability both physically and chemically.
Standardized fraction of Hibiscus flower was obtained by macerating the
flower with petroleum ether, followed by 70% ethanol solution, and
fracinationated with ethyl acetate. The fraction containing alkaloid was defined as
standardized fraction. Optimalitation of the syrup containing standardized fraction
obtained by Simplex Lattice Design software Design Expert method version 7.1.
Marker compound (alkaloid) isolation of Hibiscus flower was done by Thin Layer
Chromatrography (TLC), Vaccum Liquid Chromatography (VLC), Preparative
Thin Layer Chromatrography (PTLC). The marker was then identified according
to UV-Vis, IR, GCMS, and NMR data. The marker concentration was determined
by KLT densitometer. The syrup obtained from optimation process was tested by
in vitro for its mucolytic activity, and also tested for its physical and chemical
properties, its resistence to microbial contamination as well as respondent
tolerability.
The result of the study shows that the marker obtained contains 4 compounds,
identified as Glycine,N,N-dimethyl, methyl ester ; 2-propanamine,N,N-dimethyl ;
1,2-Ethane diamine ; N,N,-dimethyl Glycine while Glycine,N,N-dimethyl, methyl
ester and 1,2-Ethane diamine are the major components (53.83 %, 30.96 %). The
optimum formula was defined as glycerin (37.13%), sorbitol solution 70%
(49.32%), CMC-Na 0.5% (13.54%). Physical response of the optimum formula
from this study was comparable to the prediction especially on viscosity and
pouring comfortability but not in taste and acidity. Syrup formula was less stabil
in 4 weeks of storage viewed in the acidity and viscosity level. Standardized syrup
showes in vitro mucolytic activity, and at 2.0% equal to acetylcystein syrup 0.1%.
The presence of marker syrup was undetectable after storing at 27oC, 40 oC, 55 oC,
and 70 oC for 4 weeks.
Key word: hibiscus flower, optimation of syrup, standardized fraction, mucolytic,
in vitro
Pendahuluan
Beberapa penyakit seperti bronkitis dan infeksi saluran nafas menghasilkan
mukus (Ikawati, 2006). Peningkatan produksi mukus terjadi pada kondisi tersebut,
dan mukus yang diproduksi sifatnya kental, sehingga hal ini berpengaruh pada

pernafasan. Secara fisiologis silia tidak mampu mengeluarkan mukus karena

terlalu kental (Hitner and Nagle, 1999). Mukus kental dapat dikeluarkan melalui
proses pengenceran. Bunga kembang sepatu (Hibiscus rosa-sinensis L.) termasuk
salah satu obat tradisional yang dipakai masyarakat sebagai peluruh dahak atau
pengencer mukus (Departemen Kesehatan, 1985).
Banyak penelitian terhadap kembang sepatu, akan tetapi penelitian mengenai
formulasi fraksi dari bunga kembang sepatu sebagai mukolitik secara in vitro
belum ada. Penelitian yang telah dilakukan antara lain Ekstrak etanolik bunga
kembang sepatu mampu menghambat pertumbuhan Mycobacterium tuberculosis
yang sensitif dan resisten (Ruban and Gajalakshmi, 2012). Ekstrak etanolik akar
kembang sepatu mempunyai aktivitas sebagai antiimplantasi (Vasudeva and
Sharma, 2008). Ekstrak petroleum eter, hidroalkohol, dan kloroform bunga
kembang sepatu mampu menurunkan tekanan darah (Siddiqui et al., 2006).
Gauthaman et al. (2006) melaporkan khasiat bunga kembang sepatu dalam
meningkatkan senyawa antioksidan endogen miokardial, sehingga berefek
kardioprotektif.
Penggunaan bunga kembang sepatu secara langsung dinilai tidak praktis,
efektif, dan acceptable. Penggunaan dalam bentuk fraksi terstandar bunga
kembang sepatu dinilai lebih efektif, sehingga perlu diformulasikan dalam sediaan
sirup. Senyawa penanda (marker) merupakan suatu senyawa yang dapat dijadikan
untuk menilai jaminan kualitas fraksi sebagai zat aktif. Dengan demikian fraksi
akan mempunyai kualitas yang konstan.

Sirup merupakan bentuk sediaan cair yang mempunyai nilai lebih antara lain
dapat digunakan oleh hampir semua usia, cepat diabsorpsi, sehingga cepat
menimbulkan efek. Fraksi mempunyai karakteristik rasa yang tidak enak dan
mengandung bagian-bagian yang tidak larut. Bahan-bahan tambahan sangat
diperlukan untuk membuat sediaan sirup yang acceptable dari fraksi bunga
kembang sepatu. Gliserin ditambahkan sebagai kosolven untuk zat aktif yang
sukar larut. Bagian fraksi yang sukar larut didispersikan dengan penambahan
CMC Na. Rasa yang tidak enak dikurangi dengan penambahan sorbitol.
Komposisi yang proporsional antara ketiga bahan tersebut perlu dicari untuk
menghasilkan sirup yang acceptable. Simplex Lattice Designe (SLD) merupakan
salah satu metoda yang sesuai untuk optimasi formula sirup fraksi dengan ketiga
komponen tersebut (Bolton, 1996). Oleh karena itu, perlu dilakukan penelitian
mengenai optimasi formula sediaan sirup mukolitik fraksi terstandar bunga
kembang sepatu (Hibiscus rosa-sinensis L.).
Adanya penelitian secara ilmiah tentang obat tradisional diharapkan
mempercepat penerimaan oleh masyarakat luas dan kalangan medis sehingga
dapat dipertanggungjawabkan secara ilmiah sekaligus membantu kelestariannya.
Metodologi Penelian
Bahan
MSD), IR (Perkin Elmer Spectrum 100), NMR (Bruker Avance 400 NMR
spectrometer Rheinstetten, Germany) dan KLT-densitometer (CAMAG TLC
Scanner 3).

Jalan Penelitian
1. Determinasi tanaman
2. Pembuatan fraksi terstandar dengan cara maserasi dengan etanol 70%
dan fraksinasi dengan etilasetat.
3. Isolasi senyawa penanda (alkaloid) dengan VLC dan KLTP dan
penetapan kadar alkaloid dengan KLT-densitometri.
4. Identifikasi senyawa dengan spektrofotometer UV-Vis, IR, GC-MS
dan NMR. Selanjutnya fraksi etanolik disebut sebagai fraksi
terstandar.
5. Uji aktivitas mukolitik secara in vitro dengan menghitung penurunan
viskositas mukus dengan larutan uji (larutan mukus dapar fosfat pH7
dan ekstrak etanolik, fraksi etanolik dan fraksi etilasetat) terhadap
larutan mukus, dengan menggunakan viskometer Ostwald.
6. Optimasi sirup fraksi terstandar dengan mengkombinasikan gliserin,
sorbitol dan CMC Na dengan menggunakan metode Simplex Lattice
Design (SLD).
7. Uji sifat fisik sirup hasil optimasi.
8. Uji stabilitas kimiawi sirup hasil optimasi dengan menggunakan suhu
27, 40, 55, dan 700C.
9. Uji aktivitas mukolitik secara in vitro sirup hasil optimasi.

Analisa Data
Data yang diperoleh (aktivitas mukolitik secara in vitro, sifat fisik sirup fraksi
terstandar, aktivitas mukolitik secara in vitro) diuji dengan Anova dan t-tes
dengan taraf kepercayaan 95%. Stabilitas kimia sirup dianalisa secara deskriptif.
Hasil dan Pembahasan
Setelah dicocokkan dengan acuan baku (Backer and Van den Brink, 1965),
maka tanaman tersebut adalah Hibiscus rosa-sinensis L.
Rendemen ekstrak yang didapat adalah sebanyak 32,09 %, kemudian rendemen
fraksi etanolik sebesar 84,50% dan fraksi etilasetat sebesar 13,91 %.
Ekstrak etanolik kadar 1,00, 1,25, dan 1,50% menunjukkan adanya
aktivitas mukolitik secara in vitro, dan pada kadar 1,00% menunjukkan aktivitas
mukolitik yang setara dengan aktivitas mukolitik asetilsistein 0,10%.
Viskositas mukus dengan adanya fraksi etanolik dari ekstrak etanol bunga
kembang sepatu menunjukkan adanya penurunan dibandingkan dengan kontrol
negatif (lebih kecil), sehingga dapat diartikan bahwa fraksi etanolik dengan kadar
0,60, 0,80 dan 1,00% mempunyai aktivitas mukolitik secara in vitro.
Setelah dianalisis dengan anova dan uji t LSD, maka didapatkan hasil
bahwa viskositas dengan variasi kadar fraksi etanolik berbeda bermakna dengan
kontrol negatif. Hal ini mempunyai arti bahwa fraksi etanolik dengan kadar 0,60,
0,80, dan 1,00% mempunyai aktivitas mukolitik dengan menurunkan mukus
secara in vitro dan setara dengan kontrol positif (asetilsistein 0,10%). Hal ini
menunjukkan bahwa fraksi etanolik lebih efektif dari pada ekstrak etanolik. Fraksi
etilasetat 0,60, 0,80, dan 1,00% mempunyai viskositas yang lebih kecil daripada

kontrol negatif, artinya fraksi etilasetat pada kadar tersebut berefek sebagai
mukolitik. Setelah diuji dengan anova dan diteruskan dengan uji t LSD hasilnya
berbeda signifikan, sehingga dapat dikatakan bahwa secara in vitro fraksi
etilasetat pada kadar tersebut dapat menurunkan viskositas mukus, walaupun tidak
ada kadar fraksi etilasetat yang mempunyai viskositas yang tidak berbeda
bermakna dengan kontrol positif (asetilsistein 0,10%) yang artinya secara in vitro
fraksi etilasetat belum ada yang mempunyai aktivitas mukolitik yang setara
dengan aktivitas mukolitik asetilsistein 0,10%.
Gambar 1 (d) terlihat hasil pemisahan yang baik. Alkaloid terpisah
dari senyawa lain serta terbentuk noda bulat panjang berwarna merah setelah
disemprot dengan pereaksi Dragendorff. Noda terletak pada hRf 13. Penggunaan
fase gerak etilasetat : metanol 1:5 menghasilkan noda yang lebih baik dibanding
dengan fase gerak yang lain (Gambar 1a, 1b, 1c, 1e).
100

hRf

90

80
70
60
50
40
30
20
10
0

(a)
(b)
(c)
(d)
(e)
Gambar 1. Hasil pemisahan dengan berbagai variasi fase gerak: (a) toluen : etilasetat :
dietilamin (7 : 2: 1); (b) etilasetat : metanol (9 : 1); (c) etilasetat : metanol (1:1); (d) etil
asetat : metanol (1:5); (e) etilasetat : metanol (1:9)

Fraksinasi dengan menggunakan metode Vacuum Liquid Chromatography

(VLC) menghasilkan 7 fraksi dan fraksi 6 dan 7 yang mengandung alkaloid
sebagai senyawa penanda dengan kadar sebanyak 0,35 0,03% yang ditentukan
dengan KLT-densitometer.
Isolat alkaloid teridentifikasi sebagai senyawa Glycine,N,N-dimethyl,
methyl ester ; 2-propanamine,N,N-dimethyl ; 1,2-Ethane diamine ; dan N,N,dimethyl Glycine. Berdasarkan atas data GC-MS komponen terbesar adalah
Glycine,N,N-dimethyl, methyl ester dan 1,2-Ethane diamine. Dalam penelitian ini
belum dapat dilakukan uji aktivitas mukolitik pada senyawa alkaloid karena
dalam isolat terdapat 4 senyawa. Dilihat dari strukturnya maka tidak ada yang
mirip dengan asetilsistein, sehingga senyawa penanda dalam bunga kembang
sepatu bukan merupakan senyawa yang berkhasiat.
Formula optimum sirup ditentukan dengan data sifat fisik tujuh formula
sirup pada minggu ke-0. Penentuan formula optimum dilakukan dengan metode
Simplex Lattice Design dengan software Design Expert versi 7.1. Karakteristik
sifat fisik sirup yang digunakan dalam penetapan formula optimum adalah
viskositas, waktu tuang, tanggaanp responden, dan pH.
Berdasarkan analisis dengan menggunakan software Design Expert versi
7.1 untuk memprediksi formula optimum sirup fraksi terstandar bunga kembang
sepatu diperoleh dihasilkan superimposed dari contour plot respon viskositas,
waktu tuang, derajat keasaman, dan tanggapan responden sirup fraksi terstandar
bunga kembang sepatu seperti yang terlihat pada gambar 2. Superimposed yang
diperoleh menunjukkan daerah yang berwarna kuning yang menunjukkan daerah

yang memberikan respon optimum. Pada daerah tersebut didapatkan satu prediksi
formula optimum dengan nilai desirability sebesar 0,994 (gambar 2).

Gambar 2. Superimposed dari contour plot respon viskositas, waktu tuang, derajat
keasaman, dan tanggapan responden sirup fraksi terstandar bunga kembang sepatu

Komposisi formula optimum yang diperoleh dari analisis menggunakan

software Design Expert versi 7.1 adalah gliserin sebesar 37,13%; larutan sorbitol
70% sebesar 49,32%; dan mucilago CMC-Na 0,5% sebesar 13,54%, Prediksi
respon yang diperoleh dari prediksi formula optimum yaitu viskositas sebesar
7,28 mPaS, waktu tuang sebesar 2,98 detik, pH sebesar 3,55, dan tanggapan
responden sebesar 3,29.
Prediksi respon yang diperoleh dari analisis menggunakan software Design
Expert versi 7.1 selanjutnya dibandingkan dengan respon yang diperoleh pada
percobaan. Formula optimal yang didapat dari software Design Expert dibuat dan
dilakukan evaluasi sifat fisik untuk dibandingkan dengan sifat fisik formula

prediksi. Analisis yang digunakan adalah one sample t-test dengan taraf
kepercayaan 95%. Berikut ini adalah hasil one sample t-test untuk viskositas, pH,
dan waktu tuang, dan tanggapan responden formula optimal (tabel 1).
Respon viskositas dan waktu tuang

berbeda tidak bermakna antara

prediksi software Design Expert versi 7.1 dengan hasil percobaan, sedangkan
respon pH dan tanggapan responden berbeda bermakna antara prediksi software
Design Expert versi 7.1 dengan hasil percobaan.
Tabel 1. Hasil Uji One Sample T-test Formula Optimal Sirup Hasil Prediksi Software
Dibandingkan dengan Hasil Percobaan

Respon

Nilai prediksi

Signifikansi

Keterangan

7,27637

Nilai
percobaan
7,33

Viskositas
(mPas)
pH

0,282

3,29053

3,423

0,000

2,97663

2,963

0,598

3,54694

3,0625

0,000

Berbeda tidak
bermakna
Berbeda
bermakna
Berbeda tidak
bermakna
Berbeda
bermakna

waktu tuang
(detik)
Tanggapan
responden

Formula optimum sirup fraksi terstandar bunga kembang sepatu, kemudian

diuji aktivitas mukolitiknya secara in vitro dilihat dari penurunan viskositas sirup
dalam larutan mukus dapar 20%. Hasilnya dapat dilihat dalam tabel 2 berikut.
Tabel 2. Aktivitas Mukolitik secara In Vitro (Viskositas) Sirup Fraksi Terstandar Bunga
Kembang Sepatu Dengan Berbagai Konsentrasi

Formula
D
E
F
G
H

Viskositas (mPaS.)
(SD)
1,780,05
1,370,05
1,280,04
1,220,03
1,140,06

Keterangan :
Formula D: sirup hasil optimasi

Formula E: sirup hasil optimasi dengan fraksi terstandar 1,20%

Formula F: sirup hasil optimasi dengan fraksi terstandar 1,60%
Formula G: sirup hasil optimasi dengan fraksi terstandar 2,00%
Formula H: sirup hasil optimasi dengan asetilsistein 0,10%

Tabel 2 menunjukkan aktivitas mukolitik secara in vitro, dalam hal ini

adalah viskositas sirup fraksi terstandar bunga Kembang Sepatu dengan konsentrasi
1,20%, 1,60%, dan 2,00% dibandingkan dengan aktivitas mukolitik sirup
asetilsistein 0,10%. Semakin tinggi konsentrasi fraksi terstandar bunga Kembang
Sepatu yang digunakan, semakin kecil viskositas sirup. Hal ini berarti bahwa
semakin banyak fraksi terstandar yang digunakan maka aktivitas mukolitik secara
in vitro semakin meningkat.
Setelah diuji dengan t-tes dengan taraf kepercayaan 95 % maka hasilnya
adalah sirup formula hasil optimasi yang mengandung fraksi terstandar kadar
1,20; 1,60; dan 2,00% mempunyai aktivitas mukolitik secara in vitro dengan
kadar fraksi terstandar 2,00% mempunyai aktivitas mukolitik setara dengan
aktivitas mukolitik sirup dengan asetilsistein 0,10% secara in vitro.
Stabilitas sirup fraksi terstandar bunga kembang sepatu diketahui juga dengan
menyimpan sirup pada suhu 270C, 400C, 550C, dan 700C selama 4 minggu.
Keberadaan alkaloid setelah sirup fraksi terstandar bunga kembang sepatu
disimpan dalam suhu 270C, 400C, 550C, dan 70 0C ditentukan dengan KLTdensitometer.
Anlisis penentuan kadar digunakan metode KLT-densitometri, yaitu dengan
melakukan scanning bercak pada 200 nm sehingga didapatkan suatu nilai
hubungan antara AUC dengan kadar seri larutan baku sehingga diperoleh suatu
persamaan regresi sebagai persamaan kurva baku Y = A+BX, untuk perhitungan

kuantitatif terhadap kadar senyawa dengan memasukkan nilai AUC yang didapat
sebagai nilai Y, pada persamaan. Namun pada senyawa alkaloid dalam sediaan
sirup fraksi ini tidak dapat ditentukan secara KLT-densitometri karena harga hRf
senyawa penanda pada fraksi yang telah diformulasikan dalam sediaan sirup
mengalami perubahan. Dilihat dari

nilai hRf yang nampak, maka senyawa

penanda mengalami peningkatan polaritas. Hasilnya dapat dilihat dalam gambar

3, sehingga dapat dikatakan bahwa suhu berpengaruh pada keberadaan alkaloid,
dengan naiknya suhu, alkaloid mengalami kerusakan. Suhu berpengaruh pada
kecepatan reaksi. Semakin tinggi temperatur, maka semakin besar tetapan
kecepatan suatu reaksi atau reaksi semakin cepat. Kenaikan suhu 100C dapat
menyebabkan kenaikan kecepatan reaksi sebesar 5,5 kali (Yoshika and Stella,
2002), sehingga penyimpanan pada suhu yang tinggi menyebabkan kerusakan
alkaloid.
Kemungkinan lain bahwa adanya komponen sirup juga berpengaruh pada
keberadaan alkaloid sebagai senyawa penanda yaitu karena kemungkinan terjadi
interaksi sehingga tidak mempunyai hRf yang sama.
Di dalam sirup terdapat asam tartrat yang merupakan asam lemah yang akan
bereaksi dengan N dari alkaloid yang bersifat basa lemah sehingga menghasilkan
garam lemah yang kelarutannya kecil, jadi tidak dapat terelusi dengan baik.

hRf
100

90
80
70
60
50
40
30
20
10
0

1 2 3 4 5
Kiri

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Kanan

Gambar 3. Hasil Penotolan Seluruh Sampel /sirup yang mengandung fraksi

terstandar (kanan 6-25), larutan baku (kiri 1-5) dengan fase diam silika
gel 60 F254 dan fase gerak etilasetat : metanol (1:5)

Kesimpulan
1.

Fraksi terstandar bunga kembang sepatu (Hibiscus rosa-sinensis L.) dengan

kadar 0,60, 0,80, dan 1,00% mempunyai aktivitas mukolitik dengan adanya
penurunan nilai viskositas larutan mukus usus sapi secara in vitro dan
mempunyai aktivitas mukolitik setara dengan aktivitas mukolitik asetilsistein
0,10%.

2. Fraksi fraksi terstandar bunga kembang sepatu mengandung senyawa alkaloid

sebagai senyawa penanda yang merupakan alkaloid golongan alifatis yang
mengandung gugus hidroksil, gugus amina, ikatan karbon rangkap dua dan
ikatan karbon rangkap tiga. Senyawa alkaloid dengan kadar 0,35 0,03%
dalam fraksi terstandar terdiri dari 4 senyawa, teridentifikasi sebagai

Glycine,N,N-dimethyl, methyl ester ; 2-propanamine,N,N-dimethyl ; 1,2Ethane diamine ; N,N,-dimethyl Glycine. Berdasarkan atas data GC-MS
komponen terbesar adalah Glycine,N,N-dimethyl, methyl ester dan 1,2-Ethane
diamine.
3. Komposisi formula optimum sirup fraksi terstandar bunga kembang sepatu
adalah gliserin sebesar 37,13%; larutan sorbitol 70% sebesar 49,32%; dan
mucilago CMC-Na 0,5% sebesar 13,54%. Formula optimum yang diperoleh
mempunyai respon viskositas dan derajat keasaman yang berbeda dengan
prediksi respon yang diberikan oleh software Design Expert versi 7.1,
sedangkan untuk respon waktu tuang dan respon tanggapan responden
menunjukkan hasil yang sama. Sirup fraksi terstandar bunga kembang sepatu
kurang stabil selama 4 minggu penyimpanan ditinjau dari respon derajat
keasaman dan waktu tuang. Setelah sirup fraksi terstandar disimpan selama 4
minggu pada suhu 270C, 400C, 550C, dan 700C, maka keberadaan alkaloid
tidak bisa dideteksi dengan KLT-densitrometer.
4. Sirup fraksi terstandar dengan kadar 1,20; 1,60; dan 2,00% mempunyai
aktivitas mukolitik secara in vitro dan sirup dengan kadar 2,00% mempunyai
aktivitas mukolitik yang sama dengan aktivitas mukolitik sirup asetilsistein
0,10%.
Daftar Acuan
Backer, C. A., dan Van den Brink, B. R. C., 1965, Flora of Java
(Spermatophytales Only), Vol. I, 3-6, 32-34, 41, 239-240, WoltrsNoordhoff, Groningen, The Netherlands.

Bolton, S., 1997, Pharmaceutical Statistics Practical and Clinical Application, 3rd
Edition, 610-619, Marcel Dekker Inc., New York.
Departemen Kesehatan, 1985, Tanaman Obat Indonesia, Jilid Pertama, 44,
Jakarta, Departemen Kesehatan Republik Indonesia.
Gauthaman, K.K., Saleem, M.T.S., Thanislas, P.T., Prabhu, V.V.,
Krishnamoorthy, K.K., Devaraj, N.S., and Somasundaram, J.S., 2006,
Cardioprotective Effect of the Hibiscus rosa sinensis Flowers in An
Oxidative Stress Model of Myocardial Ischemic Reperfusion Injury in Rat,
BMC Complementary and Alternative Medicine, 6, 32-39.
Hitner, H. and Nagle, B., 1999, Basic Pharmacology, Fourth Edition, 409,
Glencoe McGraw-Hill, New York.
Ikawati, Z., 2006, Farmakoterapi Penyakit Sistem Pernafasan, Cetakan Pertama,
27, 29-30, 32, Laboratorium Farmakoterapi dan Farmasi Klinik Bagian
Farmakologi dan Farmakoterapi Fakultas Farmasi Universitas Gadjah
Mada, Yogyakarta.
Ruban, P. And Gajalakshmi, K., 2012, In Vitro Antibacterial Activity of Hibiscus
rosa-sinensis Flower Extract Against Human Pathogens, Asian Pasific
Journal of Tropical Biomedicine, 2, 5, p. 399-403.
Siddiqui, A.A., Wani, S.M., Rajesh, R., and Alagarsamy, V., 2006, Phytochemical
and Pharmacological Investigation of Flowers of Hibiscus rosa-sinensis
Linn, Indian J. Pharm. Sci., 68 (1), 127-130.
Vasudeva, N. and Sharma, S.K., 2008, Post-Coital Antifertility Activity of
Hibiscus rosa-sinensis Linn. Roots, eCAM, 5 (1), 91-9.
Yoshioka, S. and Stella, V.J., 2002, Stability of Drugs and Dosage Forms, 30-39,
Kluwer Academic Publishers, New York.

SUMMARY
FORMULA OPTIMATION OF HIBISCUS FLOWER (Hibiscus
rosa-sinensis L.) STANDARDIZED FRACTION MUCOLYTIC
SYRUP PREPARATION IN VITRO
DISERTATION
As one of the requirement to obtain PhD degree

Proposed by:
MIMIEK MURRUKMIHADI
08/276481/SFA/00032

For
POST GRADUATE PROGRAM
PHARMACY STUDY PROGRAM
FACULTY OF PHARMACY
UNIVERSIONTY OF GADJAH MADA
YOGYAKARTA
2012

ABSTRACT
Hibiscus Flower (Hibiscus rosa-sinensis L.) has been used traditionally
against cough. It was potential as secretolytic agent, but a standardized syrup is
still needed in order to obtain optimum pharmalogycal effect. The objective of
this research was to obtain an optimum standardized fraction syrup formula of
Hibiscus flower by Simplex Lattice Design Method, as well as to determine its in
vitro mucolytic activity and stability both physically and chemically.
Standardized fraction of Hibiscus flower was obtained by macerating the
flower with petroleum ether, followed by 70% ethanol solution, and
fracinationated with ethyl acetate. The fraction containing alcaloidwas defined as
standardized fraction. Optimalitation of the syrup containing standardized fraction
obtained by Simplex Lattice Design software Design Expert method versionon
7.1. Marker compound (alkaloid) isolation of Hibiscus flower was done by Thin
Layer Chromatrography (TLC), Vaccum Liquid Chromatography (VLC),
Preparative Thin Layer Chromatrography (PTLC). The marker was then identified
according to UV-Vis, IR, GCMS, and NMR data. The marker concentration was
determined by TLC densitometer. The syrup obtained from optimation process
was tested by in vitro for its mucolytic activity, and also tested for its physical and
chemical properties, its resistence to microbial contamination as well as
respondent tolerability.
The result of the study shows that the marker obtained contains 4 compounds,
identified as Glycine,N,N-dimethyl, methyl ester ; 2-propanamine,N,N-dimethyl ;
1,2-Ethane diamine ; N,N,-dimethyl Glycine while Glycine,N,N-dimethyl, methyl
ester and 1,2-Ethane diamine are the major components (53.83 %, 30.96 %). The
optimum formula was defined as glycerin (37.13%), sorbitol solution 70%
(49.32%), CMC-Na 0.5% (13.54%). Physical response of the optimum formula
from this study was comparable to the prediction especially on viscosity and
pouring comfortability but not in taste and acidity. Syrup formula was less stabil
in 4 weeks of storage viewed in the acidity and viscosity level. Standardized syrup
showes in vitro mucolytic activity, and at 2.0% equal to acetylcystein syrup 0.1%.
The presence of marker syrup was undetectable after storing at 27oC, 40 oC, 55 oC,
and 70 oC for 4 weeks.
Key word: hibiscus flower, optimation of syrup, standardized fraction, mucolytic,
in vitro

INTRODUCTION
Some diseases such as bronchitis and respiratory tract infection produced
mucus secretion (Ikawati, 2006). The increase of mucus production occurred in
such condition and the mucus has thick consistency, which affect the breathing
function. Physiologically the cilia cannot eliminate the mucus as it was too thick
(Hitner and Nagle, 1999). The thick mucus can be eliminated through dissolution
process. Hibiscus flower (Hibiscus rosa-sinensis L.) is one of the traditional
medication utilized in the community as mucolytic agent (Ministry of Health,
1985).
Many researches had been conducted on hibiscus flower. Nevertheless,
research on fractioned formula of hibiscus flower as in vitro mucolytic agent has
not existed yet. The previous researches are, for example, ethanol extract of
hibiscus flower to inhibit the growth of antibiotic sensitive and resistant
Mycobacterium tuberculosis yang sensitif (Ruban and Gajalakshmi, 2012). Anti
implantasi activity of hibiscus flowers root ethanol extract (Vasudeva and
Sharma, 2008). The anti hipertensive properties of petroleum ether, hydro
alcohol, and chloroform of hibiscus flower extract (Siddiqui et al., 2006).
Gauthaman et al. (2006) reported the effect of hibiscus flower in increasing
endogenous anti oxiandt component in myocardium to bring cardio protective
effect.
The direct use of hibiscus flower was considered unpractical, ineffective and
unacceptable. The use of hibiscus flower in standardized fraction was considered

more effective, thus need to be formulated in syrup preparation. Marker

compound is a compound which can be utilized to assess the quality of fraction as
active ingredients. That way, fraction will have a constant quality.
Syrup is a liquid preparation which has many advantages, such as acceptable
for almost all age group, easily absorbed and having a faster effect. Fraction had
some disadvantaged characteristic such as unfavorable flavor and having parts
which cannot be dissolved. Additions of ingredients are necessary to make an
acceptable syrup preparation of hibiscus flower fraction. Glycerin was added as co
solvent for in dissolved active ingredients. The in dissolved fraction part was
dispersed with the addition of CMC Na. The flavor was improved by adding
sorbitol. Proportional composition between the three ingredients needs to be
formulated to gain an acceptable syrup preparation. Simplex Lattice Design
(SLD) was one of the appropriate methods to optimize the fraction syrup formula
containing the three components (Bolton, 1996). Therefore, a research on
optimizing the mucolytic syrup formula of standardized fraction hibiscus flower
(Hibiscus rosa-sinensis L.) needs to be conducted.
Scientific research on traditional medicines was expected to encourage
community and medical society acceptance towards traditional medicines as it has
a strong scientific basis as well as to preserve the heritage of Indonesian
traditional medicine.

Research Methodology
Means
(MSD), IR (Perkin Elmer Spectrum 100), NMR (Bruker Avance 400 NMR
spectrometer Rheinstetten, Germany) and TLC-densito meter (CAMAG TLC
Scanner 3).

Process of Reserach
1. Determination of plant species
2. Production of standardized fraction by maceration with ethanol 70%
and fractination with ethyl acetate.
3. Isolation of marker compound (alcaloid) using VLC and PTLC and
determination of alcaloid concentrationwith TLC-densitometry.
4. Identification of compound using spectrophotometer UV-Vis, IR, GCMS and NMR. Next, ethanolic fraction was refer as standardized
fraction.
5. Testing in vitro mucolytic activity by counting the decrease of mucus
viscosity using test liquid (buffer phosphate mucus liquid fosfat pH7
and ethanolic extract, ethanolic fraction and ethyl acetate fraction)
against mucus by using Ostwald viscometer.
6. Syrup optimation of standardized fraction by combining glyserin,
sorbitol and CMC Na by using Simplex Lattice Design (SLD) method.
7. Testing the physical stability of optimation syrup result.

8. Testing chemical stability of of optimation syrup result by exposing it

to temperature of 27, 40, 55, and 700C.
9. Testing the in vitro mucolytic activity of optimation syrup result.
Data Analysis
The obtained data (in vitro mucolytic activity , physical stability of standardized
fraction syrup, in vitro mucolytic activity ) was tested with Anova and t-test with
reliability of

95%. Chemical stability of of optimation syrup was analyzed

descriptively.
Result and Review
After matched with main reference (Backer and Van den Brink, 1965), the
plant was being identified as Hibiscus rosa-sinensis L.
Obtained extract was as much as 32, 09 %, followed by ethanolic fraction
as much as 84, 50% and ethyl acetate fraction as much as 13,91 %.
Ethanol extract with the concentration of 1,00, 1,25, and 1,50% shows in
vitro mucolytic activity, and at concentration of 1,00% shows mucolytic activity
equal to mucolytic activity of acetyl cystein 0,10%.
Mucus viscosity are decreasing with the presence of ethanolic fraction of
ethanol extract of hibiscus flower, if being compared to negative control (lower),
which conclude that ethanolic fraction with the concentration of 0,60, 0,80 and
1,00% had in vitro mucolytic activity .
After being analyzed with anova and t test LSD, the result was that
viscosity with varied concentration of ethanolic fraction were significantly differs,

compared to negative control. This means that ethanolic fraction with

concentration of 0, 60, 0, 80, and 1, 00% has mucolytic activity by decreasing
mucus viscosity in vitro equals to positive control (acetyl cystein 0, 10%). This
shows that ethanolic fraction is more effective than ethanol extract.
Ethyl acetate fraction of 0, 60, 0,80, and 1,00% has lower viscosity than
negative control, which means ethyl acetate fraction at that concentration level
functioned as mucolytic. After tested with anova and followed with t test LSD, the
result was significantly different, so as concluded that ethyl acetate fraction at
that level of concentration can decrease in vitro mucus viscosity, even if there are
no concentration of ethyl acetate

fraction

which has significantly different

viscosity with (acetyl cystein 0,10%), which means in vitro mucolytic activity of
ethyl acetate fraction is not equal with mucolytic activity of acetyl cystein
0,10%.
Picture 1 (d) shows result of good separation. Alcaloid was being
separated from other compounds, forming red long round stain after being
sprayed with Dragendorff reagent. The stain was located at hRf 13. The use of
moving phase of ethyl acetate : methanol 1:5 result in better staining compared to
other moving phase (picture 1a, 1b, 1c, 1e).

hRf
100

90
80
70
60
50
40
30
20
10
0

(a)
(b)
(c)
(d)
(e)
Picture 1. Separation result using varied moving phases: (a) toluene : ethyl acetate :
diethil amine (7 : 2: 1); (b) ethyl acetate : methanol (9 : 1); (c) ethyl acetate : methanol
(1:1); (d) ethyl acetate : methanol (1:5); (e) ethyl acetate : methanol (1:9)

Fractination using Vacuum Liquid Chromatography (VLC) method result

in 7 fraction and fraction 6 and 7, containing alcaloid as marker compound with
concentration level of 0,35 0,03%, determined using TLC-densito meter.
Alcaloid isolate was identified as Glycine,N,N-dimethyl, methyl ester ; 2propanamine,N,N-dimethyl ; 1,2-Ethane diamine ; and N,N,-dimethyl Glycine
compounds. Based on GC-MS data, the largest component is Glycine,N,Ndimethyl, methyl ester and 1,2-Ethane diamine. In this research, mucolytic
activity test on alcaloid compound cannot be conducted yet, for in the isolate there
are 4 compounds. Seen from the structure, none resembles acetyl cystein, thus,
marker compound in hibiscus flower was not the therapeutic agent.
Syrup optimal formula was determined by data of physical features of
seven syrup formula on week - 0. The deteremination of optimal formula was

conducted by using Simplex Lattice Design with Software Design Expert

versionon 7.1. characteristic of physical features of the syrup used in optimal
formula determination is viscosity, pouring time, respondent opinion, and pH.
Based on analysis using Software Design Expert versionon 7.1 to predict
optimal formula of standardized fraction syrup of hibiscus flower, we obtained
superimposed result of contour plot on viscosity response, pouring time, acidity
level, and respondent opinion on standardized fraction syrup of hibiscus flower as
seen on picture 2. The obtained superimposed showed yellow parts which refers
to parts of optimal response. In that part we obtained a prediction of optimum
formula with desirability value of 0,994 (picture 2).

Picture 2. Superimposed result of contour plot on viscosity response, pouring time, acidity level, and
respondent opinion on standardized fraction syrup of hibiscus flower

Composition of optimal formula, obtained from analysis using Software

Design Expert version 7.1 are glycerine as much as 37,13%; sorbitol solution
70% as much as 49,32%; and mucilago CMC-Na 0,5% as much as 13,54%.
Response prediction obtained from optimal formula prediction was viscosity as

much as 7,28 mPaS, pouring time as much as 2,98 second, pH as much as 3,55,
and respondent opinion as much as 3,29.
Response prediction obtained from analysis using Software Design
Expert version 7.1 was then compared to response obtained from the research.
Optimal formula obtained from Software Design Expert was produced and
evaluated for its physical features to be compared with physical features of
prediction formula. Analysis that was being used is one sample t-test with
reliability level of 95%. The following are the result of one sample t-test for
viscosity, pH, pouring time, and Respondent opinion on formula optimal (table 1).
Viscosity response and pouring time shows no significant difference
between software Design Expert version 7.1 prediction with research result,
whereas pH response and Respondent opinion differ significantly between
software Design Expert version 7.1 prediction and the research result.
Table 1. Test result of One Sample T-test Sirup Optimal Formula, Software prediction result
compared to research result

Response
Viscosity
(mPas)
pH
Pouring time
(detik)
Respondent
opinion

Prediction
result
7,27637

Research
result
7,33

Significance

Conclusion

0,282

3,29053

3,423

0,000

2,97663

2,963

0,598

3,54694

3,0625

0,000

No significant
difference
Significantly
different
No significant
difference
Significantly
different

Optimal formula of hibiscus flower standardized fraction syrup was then

tested for its in vitro mucolytic activity, assessed from the decrease of syrup

viscosity in buffer mucus solution 20%. The result can be seen in the following
table 2.
Table 2. In vitro mucolytic activity (Viscosity) of hibiscus flower standardized fraction syrup
formula with various concentration levels

Formula
D
E
F
G
H

Viscosity (mPaS.)
(SD)
1,780,05
1,370,05
1,280,04
1,220,03
1,140,06

Information:
Formula D: optimized syrup
Formula E: optimized syrup with standardized fraction of 1,20%
Formula F: optimized syrup with standardized fraction of 1,60%
Formula G: optimized syrup with standardized fraction of 2,00%
Formula H: optimized syrup with acetyl cystein 0,10%

Table 2 shows in vitro mucolytic activity, in this case was the viscosity of
standardized fraction hibiscus flower syrup with the concentration of 1,20%,
1,60%, and 2,00% compared with the mucolytic activity of acetyl cystein syrup
0,10%. The higher concentration level of standardized fraction hibiscus flower
syrup being used will cause the lower viscosity of the syrup. This shows that the
more standardized fraction being used will increase the in vitro mucolytic activity.
After being tested using t-test with reliability level of 95 %, the result is
optimized formula syrup containing standardized fraction with concentration level
of 1, 20; 1, 60; and 2 ,00% has in vitro mucolytic activity. Standardized fraction
with concentration level of 2,00% has in vitro mucolytic activity equal to in vitro
mucolytic activity of acetyl cystein 0,10%.
Stability of standardized fraction hibiscus flower sirup was discovered after
storage of syrup at the temperature of 270C, 400C, 550C, and 700C for 4 weeks.

Alcaloid presence after standardized fraction hibiscus flower syrup was stored in
temperature of 27 0C, 40 0C, 55 0C, and 70 0C was determined with TLCdensitometer.
Analysis of concentration level determination using TLC-densitometri
method, which was by conducting scanning on stains on 200 nm so as obtained
a related value between AUC with main solution serial concentration level to
obtain a regression function as main curve function Y = A+BX, for quantitative
measurement on compounds concentration level by inserting AUC value which
was obtained as Y value, on the function. However, the alcaloid compound in this
fraction syrup preparation can not be determined with TLC-densitometri for the
value of hRf in the marker compound on the formulated fraction in the syrup
preparation had altered. Seen from the emerging hRf value, the marker compound
had a polarity increase. Which can be seen in picture 3, so as to say that
temperature can affect the presence of the alkaloid, with the temperature increase,
alcaloid will be destroyed. Temperature also affects reaction velocity. The higher
the temperature, the higher the constant for reaction velocity or the reaction will
go faster. The increase of 100C will cause the increase of reaction velocity as
much as 5, 5 times (Yoshika and Stella, 2002). Hence, storage at high temperature
will cause alcaloid damage.
Other possibility is that the presence of syrup components will also affect the
presence of alcaloid as marker compound due to the possibility of chemical
interaction so the hRf value will not be the same.

The syrup contains tartrate acid, a weak acid which will react with N of the
alcaloid which is a weak base and resulting in weak salts of low solubility, so it
cannot be finely elucidate.

hRf
100

90
80
70
60
50
40
30
20
10
0

1 2 3 4 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Left

Right

Picture 3. Result of pointing of all syrup simple containing standardized fraction

(right: 6-25), main solution (left: 1-5) with static phase of silica gel 60
F254 and moving phase of ethyl acetate: methanol (1:5)

Conclusion
1.

Standardized fraction syrup of hibiscus flower (Hibiscus rosa-sinensis L.)

with concentration level of 0,60, 0,80, and 1,00% has in vitro mucolytic
activity

with the decrease of viscosity value of mucus liquid in cows

intestine and has the mucolytic activity equal to acetyl cystein 0,10%.
2. Standardized fraction syrup of hibiscus flower has alcaloid compound as
marker compound which was an alyphatic alcaloid containing hidroxyl and

amine bond, double and triple strand carbon bond. Alcaloid compound with
the concentration level of 0,35 0,03% in the standardized fraction consist of
4 compound, identified as Glycine,N,N-dimethyl, methyl ester ; 2propanamine,N,N-dimethyl ; 1,2-Ethane diamine ; and N,N,-dimethyl
Glycine. Based on GC-MS data, the largest components are Glycine,N,Ndimethyl, methyl ester and 1,2-Ethane diamine.
3. Composition of optimal formula standardized fraction hibiscus flower syrup
are glycerine as much as 37,13%; sorbitol solution 70% as much as 49,32%;
and mucilago CMC-Na 0,5% as much as 13,54%. Optimal formula obtained
has viscosity response and acidity level which was different to response
prediction given by Software Design Expert version 7.1, whereas for the
pouring time and respondent opinion shows the similar result. Standardized
fraction syrup of hibiscus flower is less stable in 4 weeks of storage assessed
from the acidity level and pouring time. After the standardized fraction syrup
was being stored for 4 weeks in temperature of 270C, 400C, 550C, and 700C,
the alcaloid presence cannot be detected with TLC-densitrometer.
4. Standardized fraction syrup with concentration level of 1,20; 1,60; and 2,00%
has in vitro mucolytic activity and syrup with concentration level of 2,00%
has equal mucolytic activity to acetyl cystein syrup 0,10%.

Reference
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(Spermatophytales Only), Vol. I, 3-6, 32-34, 41, 239-240, WoltrsNoordhoff, Groningen, The Netherlands.
Bolton, S., 1997, Pharmaceutical Statistics Practical and Clinical Application, 3rd
Edition, 610-619, Marcel Dekker Inc., New York.
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Ministry of Health Republic of Indonesia.
Gauthaman, K.K., Saleem, M.T.S., Thanislas, P.T., Prabhu, V.V.,
Krishnamoorthy, K.K., Devaraj, N.S., and Somasundaram, J.S., 2006,
Cardioprotective Effect of the Hibiscus rosa sinensis Flowers in An
Oxidative Stress Model of Myocardial Ischemic Reperfusion Injury in Rat,
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Hitner, H. and Nagle, B., 1999, Basic Pharmacology, Fourth Edition, 409,
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Hibiscus rosa-sinensis Linn. Roots, eCAM, 5 (1), 91-9.
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