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).

(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)

0

20

30

40

50

60

70

80

90

100

10

hRf

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 Nilai percobaan

Signifikansi Keterangan

Viskositas (mPas)

7,27637 7,33 0,282 Berbeda tidak bermakna

pH 3,29053 3,423 0,000 Berbeda bermakna

waktu tuang (detik)

2,97663 2,963 0,598 Berbeda tidak bermakna

Tanggapan responden

3,54694 3,0625 0,000 Berbeda bermakna

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 Viskositas (mPaS.)

(±SD) D 1,78±0,05 E 1,37±0,05 F 1,28±0,04 G 1,22±0,03 H 1,14±0,06

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 KLT-

densitometer.

Análisis penentuan kadar digunakan metode KLT-densitometri, yaitu dengan

melakukan scanning bercak pada λ 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

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

Kiri 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

0

20

30

40

50

60

70

80

90

100

10

Glycine,N,N-dimethyl, methyl ester ; 2-propanamine,N,N-dimethyl ; 1,2-

Ethane 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, Wolt’rs-Noordhoff, 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 flower’s 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, GC-

MS 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).

(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 ; 2-

propanamine,N,N-dimethyl ; 1,2-Ethane diamine ; and N,N,-dimethyl Glycine

compounds. Based on GC-MS data, the largest component is Glycine,N,N-

dimethyl, 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

0

20

30

40

50

60

70

80

90

100

10

hRf

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 Prediction result

Research result

Significance Conclusion

Viscosity (mPas)

7,27637 7,33 0,282 No significant difference

pH 3,29053 3,423 0,000 Significantly different

Pouring time (detik)

2,97663 2,963 0,598 No significant difference

Respondent opinion

3,54694 3,0625 0,000 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 Viscosity (mPaS.)

(±SD) D 1,78±0,05 E 1,37±0,05 F 1,28±0,04 G 1,22±0,03 H 1,14±0,06

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 TLC-

densitometer.

Analysis of concentration level determination using TLC-densitometri

method, which was by conducting scanning on stains on λ 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 compound’s 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.

hRf

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.

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 cow’s

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

0

20

30

40

50

60

70

80

90

100

10

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 ; 2-

propanamine,N,N-dimethyl ; 1,2-Ethane diamine ; and N,N,-dimethyl

Glycine. Based on GC-MS data, the largest components are Glycine,N,N-

dimethyl, 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

Backer, C. A., and Van den Brink, B. R. C., 1965, Flora of Java (Spermatophytales Only), Vol. I, 3-6, 32-34, 41, 239-240, Wolt’rs-Noordhoff, Groningen, The Netherlands.

Bolton, S., 1997, Pharmaceutical Statistics Practical and Clinical Application, 3rd Edition, 610-619, Marcel Dekker Inc., New York.

Ministry of Health, 1985, Indonesian Medicine Herbs, First edition, 44, Jakarta, 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, 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, Pharmacotherapy of Respiratory System Disease, First Edition,

27, 29-30, 32, Pharmacotherapy Laboratorium and Clinical Pharmacy of Pharmacology and Pharmacotherapy Faculty of Pharmacy University of 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.