Trad. Med. J.

, January - April 2016 Submitted : 22-01-2016

Vol. 21(1), p 6-11 Revised : 24-03-2016
ISSN : 1410-5918 Accepted : 01-04-2016

QUALITY STANDARDIZATION OF BROTOWALI (Tinospora crispa)

STEM EXTRACT
STANDARDISASI KUALITAS EKSTRAK BATANG BROTOWALI (Tinospora crispa)
Harwoko1* and Nur Amalia Choironi1
Laboratory of Pharmaceutical Biology, Faculty of Health Sciences, Universitas Jenderal Soedirman 1
Jl. dr. Soeparno Karangwangkal Purwokerto 53123

ABSTRACT

Brotowali (Tinospora crispa) has been traditionally used for the treatment of gout and scientifically
reported as analgesic, anti-inflammatory, and antihyperuricemic agents. Tinospora crispa stem is one of
herbal medicine material that its quality should be standardized. This study aims to determine the quality
parameters of the T. crispa ethanolic extract included specific and non-specific parameters. Brotowali stem
were macerated using ethanol 70%, then the non-specific parameters such as the water content, total ash,
total contaminant number of bacteria and fungus were determined. The specific parameters including
organoleptic properties, water soluble extract, ethanol soluble extract, and the thin layer chromatography
(TLC) profile have also been determined. The parameter values were compared to the qualification of
traditional medicine from Department of Health (Depkes R.I.). The result showed that T.crispa stem ethanolic
extract has the water content was 8.120.06% and the total ash was 5.20 0.12%. The microbiology results
showed that the total contaminant of bacteria as much as 5 x 102 CFU/g and fungus as much as 5 x 103
CFU/g. This extract was brown viscous extract, bitter taste and characteristic odor with water soluble
fraction was 45.09 0.67% and ethanol soluble fraction was 14.19 0.14%. The TLC profile of ethanolic
extract indicates the existence of flavonoids and alkaloids. Total flavonoids of brotowali extract (32.65
0.20%) rutin equivalent.
Key words: Tinospora crispa, brotowali, quality standardization, standardized extract

ABSTRAK

Brotowali (Tinospora crispa) secara tradisional telah digunakan untuk pengobatan asam urat dan
secara ilmiah telah dilaporkan sebagai analgesik, antiinflamasi, dan antihiperurisemi. Batang brotowali
termasuk salah satu bahan jamu yang perlu dilakukan standardisasi mutu. Penelitian ini bertujuan untuk
menetapkan parameter mutu ekstrak etanolik batang brotowali yang meliputi parameter umum dan
spesifik. Ekstrak batang brotowali dibuat dengan metode maserasi menggunakan etanol 70% selama 3 x 24
jam. Parameter umum yang ditetapkan meliputi kadar air, kadar abu total, angka lempeng total, dan angka
kapang, sedangkan parameter spesifik seperti organoleptik, kadar sari larut air dan etanol serta profil
kromatografi lapis tipis juga ditentukan. Nilai parameter yang diperoleh dibandingkan dengan pedoman
standardisasi mutu ekstrak tumbuhan obat. Hasil penelitian menunjukkan bahwa ekstrak memiliki kadar air
sebesar 8,120,06% dan kadar abu total 5,200,12%, sedangkan angka lempeng total 5x10 2 CFU/g dan
angka kapang 5x103 CFU/g. Ekstrak etanolik batang brotowali memiliki karakteristik berupa ekstrak kental
berwarna coklat tua, berasa pahit dan berbau khas dengan kadar sari larut air sebesar 45,090,67% dan
kadar sari larut dalam etanol sebesar 14,190,14%. Selain itu, profil kromatografi lapis tipis ekstrak
etanolik menunjukkan adanya senyawa alkaloid dan flavonoid. Ekstrak ini memiliki kandungan total
flavonoid sebesar 3,710,05% setara dengan rutin.
Kata kunci: Tinospora crispa, brotowali, standardisasi kualitas, ekstrak terstandar

INTRODUCTION and more than 1,000 species of medicinal plants

Indonesia is a second largest biodiversity grow in Indonesia have been used in traditional
country that provides many traditional medicines medicine industries. The number of these
for various diseases. Over 30,000 species of plants industries especially home scale (IKOT) increased
significantly from 907 in 2002 to 1,413 in 2010
Corresponding Author : Harwoko (Wahyuningsih, 2006; Dewoto, 2007). Most of
Email : [email protected] traditional medicine products were prepared in

Traditional Medicine Journal, 21(1), 2016 6

 QUALITY STANDARDIZATION OF BROTOWALI

the form of extract. The kinds of extract were Indonesia. It was throughly washed, wet sortation,
viscous extract, dry extract, and liquid extract that dried, and grinded into powder. One kilogram
produced according to the active constituent and sample were extracted by maceration using
the dosage forms, such as capsule, tablet, liquid, ethanol 70% (in a 1: 5 ratio) for 24 hours,
pill, and etc. The extract should be standardized to subsequently filtered. Residue was re-extracted
ensure the quality and safety (Hariyati, 2005). twice with the same method and solvent. Ethanol
Brotowali (T. crispa) is well known as a extract were concentrated using rotary vacuum
bitter medicinal plant but it has various efficacy evaporator at 80C and followed by using
and has been empirically used to treat waterbath.
rheumatism, gout, bruise, and fever, also to
stimulate appetite (Dalimartha, 2008). Chemical Determination of non-specific parameters of
compounds of brotowali were reported as extract
columbine, tinocrisposide, quaternary alkaloids, Physical evaluation of extract was
saponins, tannins, polyphenols, glycosides, and conducted on water content and total ash value by
flavonoids (Sudarsono et al., 2006; Handayani, gravimetric method based on the Indonesian
2010). The antioxidant activity of brotowali stem Herbal Pharmacopeae (Ministry of Health, 2012).
according to the method used by Irianti et al. While the contaminants of total bacteria and total
(2011). The others studies also showed that T. fungus were determined by total plate count
crispa stem extract have analgetic (Sulaiman et al., method with three times of replication
2008) and anti-inflammatory effect (Hipol et al., (Department of Health, 2000).
2012). Coss et al. (1998) reported that flavonoids
and alkaloids could be correlated to xanthine Determination of specific parameters of
oxidase inhibitor activity. It is can inhibit extract
production of uric acid, an endogenous substance The specific parameters included organo-
involved gout disease. leptic, water soluble extract, ethanol soluble
Brotowali has the potential compounds to extract, the phytochemical properties, and total
be developed as a raw material of standardized flavonoid content. The organoleptic of brotowali
herbal medicine or phytopharmaca, especially for extract include colour, odor, flavour and the
antihyperuricemia (anti gout). Raw material of consistency. Determination of water soluble
extract which will be developed as a standardized extract and ethanol soluble extract was conducted
herbal medicine needed standardization process. based on the Indonesian Herbal Pharmacopeae
Accordingly, this study about standardization (Ministry of Health, 2012). The phytochemical of
of brotowali ethanolic extract was aimed brotowali extract was identified by TLC method.
to determine the quality parameters of raw Total flavonoid content was determined based on
materials included specific and non-specific modified colorimetric method of Chang et al.
parameters. (2002) using rutin as a reference standard.

METHODOLOGY Data analysis

Materials The data were descriptively analysed
Stem of brotowali (T. crispa) used in this according to the guidebook about quality
research was collected from two different areas standardization of extract from Department of
that are Sumbang, Banyumas and Buayan, Health Republic of Indonesia (Depkes R.I, 2000).
Kebumen, Central Java, Indonesia. The plant was
authenticated at Laboratory of Plant Taxonomy, RESULTS AND DISCUSSION
Faculty of Biology, Universitas Jenderal In the present study, extraction of one kg T.
Soedirman. The voucher specimen was stored in a crispa dry stem with 70% ethanol yielded 193.4 g
herbarium of the Laboratory of Pharmaceutical of a viscous ethanolic extract (19.34%) which is
Biology, Universitas Jenderal Soedirman. The more than the rendement of 96% ethanolic extract
chemicals included ethanol 70%, TLC plate silica reported by Irianti et al. (2011) only 12.02%.
gel 60 F254 (Merck, Germany), Dragendorff; However, this result is less than the rendement of
citroboric reagent, rutin, Nutrient Agar/NA 96% ethanolic extract was 20.25% (Mutiatikum et
(Merck, Germany) and Potato Dextrose Agar/PDA al, 2004). Accordingly, the higher polarity of the
(Merck, Germany). solvent, the more yield of extract.
Quality standardization of brotowali extract
Preparation and extraction of sample was determined by non-specific and specific para-
Stem of brotowali was selected from meters. The water content was measured by
Sumbang district, Banyumas regency, Central Java, gravimetric method, while the microbial conta-

7 Traditional Medicine Journal, 21(1), 2016

Harwoko

Table I. Non-specific parameters of brotowali ethanolic extract

Parameters Measured values Quality standard for extract
Water content 7.8 1.9% 10%
Total ash 4.75 0.25% 5%
Total contaminant of bacteria 1 x 104 CFU/g < 106 CFU/g
Total contaminant of fungus 0.33 x 104 CFU/g < 104 CFU/g

Table II. Percentage of total flavonoid content in ethanolic extract of T. crispa stem
Sample concentrationa Absorbance (n) Total flavonoid in Total flavonoid
(ppm) ( 415 nm) each sampleb (ppm) contentc (RE % b/b)
0.314 129.0 32.25
400
0.321 131.8 32.95
0.319 131.0 32.75
Mean SEM 32.65 0.20

b
Explanation : RE = Rutin Equivalent: c= x 100%
a

VISIBLE UV366
before sprayed by after sprayed by before sprayed by after sprayed by
Dragendorf reagent Dragendorf reagent citroboric reagent citroboric reagent

Figure 1. TLC profile of brotowali ethanolic extract (1) and rutin (2) on silica gel 60 F254 plate as stationary
phase and chloroform: methanol (9:1) (A); BAW (4:1:5) (B) as mobile phase

minations such as total bacteria and fungus Non-specific parameters of brotowali

number were determined by microbiological ethanolic extract were shown in table I. The
testing. Several factors determine the values of each parameter does not exceed the
microbiological quality of medicinal plants maximum limits or ranges were allowed by the
included plant composition (antimicrobial requirements of a guidebook. However, non-
compounds) as intrinsic factors, and also extrinsic specific parameters was determined for brotowali
factors such as location, post-harvesting, and ethanolic extract had been asserted complying
exogenous microbial contaminations (Kneifel et with the quality standard. These parameters
al., 2002). showed correlated to purity and contamination in
that extract (Department of Health, 2000).

Traditional Medicine Journal, 21(1), 2016 8

 QUALITY STANDARDIZATION OF BROTOWALI

Figure 2. Linear curve of rutin concentration (ppm) versus absorbance for determination of total flavonoid
content in T. crispa stem ethanolic extract

Organoleptic examination showed that glycoside, genkwanin 7-glycoside, luteolin 4'-

brotowali ethanolic extract has brown methyl ether 3'-glycosides, diosmetin and
viscous extract, bitter taste, and specific genkwanin (Cotelle, 2001), catechin, luteolin,
odor. Determination of water soluble fraction morin, and rutin (Amom et al., 2009).
(45.0870.636%) had highest solubility than Total flavonoid content was determined by
ethanol soluble fraction (14.194 0.143%). colorimetric method according to Chang et al.
The result indicated that brotowali ethanolic (2002) using rutin as a reference standard.
extract contain mostly polar compounds. Principally, the procedure is related to the
Brotowali ethanolic extract exhibited the presence formation of complex between flavonoid and AlCl 3
of alkaloids and flavonoids based on the TLC that produces a yellow coloured solution. The
profile (Fig. 1). The positive result of alkaloids was absorbance was measured by spectrophotometer
characterized by the appearance of orange color UV-Vis at maximum wavelength of 415 nm. The
after sprayed by Dragendorff reagent (Fig. 1A) absorbances of concentration series of quercetin
(Harborne, 1996). were plotted to their concentration to yield a
Based on the chromatogram on Figure 1B, linear calibration curve of rutin (y = 0.0026x -
brotowali ethanolic extract exhibited a clear 0.023) with coefficient of correlation (r2) value of
separation when developed using a mobile phase 0.992 (Figure 2). In this study, total flavonoid
n-buthanol-glacial acetic acid-water/BAW (4:1:5 content of brotowali extract was 32.650.20%. It
v/v, upper phase). The TLC profile showed some means that each 100 g dry weight of ethanolic
spots that have hRf values of 10; 44; 52; 62; 70 extract contained total flavonoid equivalent to 33g
with brownish yellow fluorescens under UV366 of rutin.
after sprayed by citroboric reagent. Moreover, In the present study, ethanolic extract of
rutin spot as a reference also showed a yellowish brotowali stem showed high content of total
brown fluorescens at hRf 60 under UV366 after flavonoids. Rutin is one kind of flavonoid
sprayed by this reagent. TLC profile of extract compounds in brotowali, but there are another
showed the hRf values 5-20 and 70-80 whose flavonoids like apigenin and luteolin. Reportedly,
yellow fluorescence showed higher intensity of these flavonoids exhibited antihyperuricemic
flavonoid detected by UV366 (Fig. 1B). The activity due to their potential effect on lowering
flavonoid type which expected are flavonols uric acid level (Chen et al., 2011; de Souza et al.,
without free 5-OH group or flavonols with 2012). Brotowali extract whose flavonoids content
unsubstituted 5-OH group (Wagner and Bladt, also responsible for antioxidant (Irianti et al.,
1996). Flavonoids types contained in brotowali 2011) and antihyperuricemic activity (Harwoko et
which were previously reported such as O- al., 2015). Antioxidant activity naturally occurring
glycoside flavonoids (apigenin) and flavone in plants was expected to limit microbial
glycosides, namely luteolin 4'- methyl ether 7-

9 Traditional Medicine Journal, 21(1), 2016

Harwoko

contaminant (Mansour and Khalil, 2000). Department of Health. 1978. Materia Medika
However, medicinal plants as material of herbal Indonesia. 2nd Edition. Departemen
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Departemen Kesehatan Republik Indonesia,
CONCLUSION Jakarta. pp 9-12.
Ethanolic extract of T. crispa stem showed De Souza, M.R., de Paula, C.A., de Resende, M.L.P.,
the general standardization parameters i.e the Grabe-Guimaraes, A., Filho, J.D.S., and
water content 7.81.9%; total ash content Saude-Guimaraes, D.A. 2012.
4.750.25%; total contaminant of bacteria and Pharmacological basis for use of
fungus less than 104 CFU/g, respectively. In Lychnophora trichocarpha in gouty
addition, the specific parameters included water arthritis: Anti-hyperuricemic and anti-
soluble fraction 45.090.67% and ethanol soluble inflammatory effects of its extract, fraction
fraction 14.190.14%, as well as the total and constituents. Journal of
flavonoids content was 32.650.20% equivalent to Ethnopharmacology. 145: 845-850.
rutin. Dewoto, H.R. 2007. Pengembangan Obat
Tradisional Indonesia Menjadi Fitofarmaka.
ACKNOWLEDGEMENT Majalah Kedokteran Indonesia. 57 (7): 205-
We thank to Universitas Jenderal 211.
Soedirman for institutional research grants and Handayani. 2010. Efek Antiangiogenik Ekstrak
also the Rifka Husniati as laboratory assistant and Kloroform Batang Tinospora crispa pada
Agung Prabowo as technician for helping this Membran Korio Alantoin Embrio Ayam
research. Terinduksi bFGF. Indonesian Journal of
Pharmacy. 2 (1): 124-128.
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