SUPPLEMENTARY MATERIAL
Wound healing activity of an oligomer of Alkanin / Shikonin, isolated from root bark of Onosma echioides
Gawand Nikitaa , Purnima Viveka & Gadgoli Chhaya a*
a
Saraswathi Vidya Bhavan’s College of Pharmacy, Kalyan Shil Road, Sankaranagar , Sonarpada, Dombivli (E)
421203, Dist: Thane, State : Maharashtra; India
* Saraswathi Saraswathi Vidya Bhavan’s College of Pharmacy, Kalyan Shil Road, Sankaranagar , Sonarpada,
Dombivli (E) 421203, Dist: Thane, State : Maharashtra; India
Email: chhayahg@rediffmail.com; phone 919833590156
Abstract
Root bark of Onosma echioides belonging to family Boraginaceae are reported to be rich in naphthaquinones like
alkanins and shikonins. In the present study, a dimer of alkanin / shikonin was isolated from petroleum ether (6080o C) extract of the bark and the structure of the same was elucidated through spectral studies (UV, IR, NMR, MS
and DEPT). The petroleum ether extract was found to contain 62.4% w/w of the dimer of alkanin/ shikonin and the
compound is found to promote wound healing process, when studied in the excision and incision wound models in
albino rats.
Key Words
Onosma echioides , Boraginacae, alkanin/ shikonin oligomer . Excision Wound, Incision Wound,
1
Experimental
1.1 Plant Material
Dried root bark of O. echioides was procured from the local market of Crude Drugs in Mumbai (India) and was
authenticated at G.N.Khalsa College, Mumbai, India. The voucher specimen (PYC # 261211) is also deposited in the
institute.
1.2 Extraction and Isolation of Naphthaquinone dimer (OE1)
Dried root bark of O. echioides was powdered in mixer and the powdered crude drug (100 g) was extracted with
petroleum ether (60-80o C) using Soxhlet Extraction apparatus for 18-20 hours. The extract was concentrated in
rotary flash evaporator till syrupy mass was obtained and the syrupy mass was then heated in vacuum oven at 4050o c till dryness to get the extract (5.7 gm).
The extract was loaded on column containing silica gel (60-120#,Merck) and eluted isocratically with Toluene:
Formic acid (99:1) at the flow rate of 1 ml /min. A fixed volume of the eluent (5 ml) was collected in different test
tubes and Thin Layer Chromatographic Analysis of the eluents was carried out using precoated silica gel 60 F254
(Merck) plates and Toluene: Formic acid (99:1) as mobile phase. The plates were visualized in day light for
detection of spots. All the eluents indicating single spot with Rf value of 0.43 were pooled together and dried
under vacuum to get the phytoconstituent (OE1). The compound OE1 was recrystallized using petroleum ether
(60-800C) and characterized through spectral studies.
The isolated compound OE1 was characterized through spectral studies which included UV, FT-IR, NMR (1H & 13 C),
13
C DEPT (Destortionless Enhancement by Polarization Transfer), and MS.
The FT-IR Spectrum was recorded on Jasco 4100 through preparation of KBR pallet. Mass spectrum was recorded
using Fast Atom Bombardment Mode at Central Drug Research Institute, Lucknow, India. 1H and 13 C NMR spectra
were obtained on Brucker AMX-500 spectrometer operating at 500.1 and 125.4 MHz for the two nuclei
respectively in CDCl3.13C DEPT 1350 spectrum was obtained in CDCl3 at Tata Institute of Fundamental Research ,
Mumbai , India. The quantitative elemental analysis of OE1 was carried out at SITEC Labs Pvt. Ltd. Vikhroli,
Mumbai, India.
OE 1 (I): Color: Red, solid. m.p. 79-800c; UV : λmax : 504,520,540nm; 1H NMR ( ppm) and 13 C NMR (( ppm)values
for (I) are presented in Table 1;MS (FAB )M+ : 684; Base peak: 271; FT-IR(KBr) cm-1 : 3509.81 (O-H), 2923.56 (C-H
stretching), 1729.83 (O-C=O), 1612.2 (C=C), 1455 (O-H bending),1224.58 (C-O stretching), 758.85 (C-H bending); C
(70.8%), H(6.0%), O (21.4%).
As per the literature, dimeric OH will vibrate at higher frequency than monomeric OH that is 3429 cm -1 due to
involvement of intra-molecular Hydrogen bonding. Such OH groups are reported (Assimopoulou AN et al, 2007 &
Spyros A et al 2007) to vibrate at 3550-3200 cm-1. As the OH group vibration is observed to be at 3509 cm-1,
indicates the presence oligomeric alkanin. The 13C DEPT 1350 spectrum revealed presence of six methylene groups
(C-2”, C-12, C-12’, C-13,C-13’, C-3”) , as the signals for these appeared as negative absorption. the cross proton
signals in 2-D COSY (CDCl3) spectrum indicated connectivity of protons as 11-12-13-14,11’-12’-13’-14’and 2”-3”-4”5”-6”. Thus the presence of the side chain in the structure of naphthoquinone dimer (OE1) Fig 1 was established.
The compound is reported for the first time in the root bark of O. echioides.
1.3
Quantification of OE1 in Pet Ether Extract
The compound OE1 was dissolved in petroleum ether (60-80o C) to get the concentration of 1000 ppm and
volumes of 2, 4, 6, 8 and 10 µl were applied on the silica gel GF 254HPTLC plate. 10 µl of the solution of PEOE
(1000 ppm ) in Pet Ether was applied on HPTLC plate in triplicate and the plates were developed using Toluene :
Formic acid (99:1) as mobile phase. After development the plates were scanned in Camag Scanner 3 at 520 nm.
The concentration of OE1 in PEOE was extrapolated from the standard curve.
The HPTLC analysis of content of OE 1 ( an oligomer or dimer of naphthoquinone ) in the petroleum ether extract
root bark of O. echioides was determined using HPTLC technique on silica gel stationary phase and Toluene : formic
acid (99:1) as mobile phase.(Fig. 2). The analysis revealed that the content of OE 1 in PEOE was found to be 62.4%
W/W.
1.4
Animals
For evaluation of wound healing activity of the petroleum ether extract (PEOE) and OE1, Wistar rats of either sex
(150-200 gm), were utilized. The animals were housed under standard conditions of temperature (25 + 2o C & RH
60+ 5 %) with light and dark cycle of 12 hr. The animals were fed with standard diet and water ad libitum. The
protocols were approved by the Institutional Animal Ethics Committee (Registration no. 704/CPCSEA, India dated
25/8/2003)
1.5
Pharmacological evaluation
1.5.1 Acute Dermal Toxicity Studies
The acute dermal toxicity studies were carried out as per the guidelines of OECD 402. The ointment formulations
were prepared by incorporating 0.125, 0.25 and 0 .50 gm of the petroleum ether extract and 0.0625 gm of OE1 in
simple ointment base B.P. (Wool fat 5%; Hard Paraffin 5%; Cetosteryl alcohol 5%; White Soft Paraffin 85% w/w)
quantity sufficient to make totally 25 gm. The animals were divided into three groups namely control (Simple
ointment base), test 1 (PEOE) and test 2 (OE1) with six animals in each group.
The animals were shaved at the back and the respective preparations were applied on about 1 mm 2 area and the
area was covered with cotton gauze which was secured by hypoallergic adhesive tape. At the end of 24 hours the
patches were removed and skin was observed for any visible changes such as erythema or oedema.
1.5.2
Excision wound model
The animals were divided in three groups viz. control, test and standard. A circular about 500 sq. mm was inflicted
on the dorsal thoracic region of anaesthetized rats. The control group was treated with the simple ointment base ,
the standard group was treated with the Betadine ® (Wincare Pharmaceuticals,India) ointment containing povidone
iodine ( 5 % w/w) and the test groups were treated with about 150 mg of the PEOE (0.5,1 and 2% w/w) and OE1
(0.25% W/W) containing ointments, by topical application on the wound twice a day. The treatment was
commenced from the day of infliction of wound till complete epithelization.
Wound healing activity was evaluated by determining wound closure through tracing the wound area on day 4,
6,8,10,12,14,16 and period of epithelization. The period of epithelization was calculated as the number of days
required for falling of eschar. The % epithelization or contraction of wound was calculated by the following
equation (Morton, J J and Malone, M H, 1972.).
% of wound closure =
wound area on day 0 −wound area on day n
× 100
wound area on day 0
where n =number of days 4th 6 th 8th, 12th, and 16th day.
1.5.3
Estimation of Hydroxyproline content
Excision wounds were inflicted and treated as described in 4.5.2 for ten days. On day 11, the scab was removed
and dried in oven. The dried scab was treated with concentrated Hydrochloric acid and utilized further for
determination of Hydroxyproline. The content of hydroxyproline was determined by following the protocols
described by (Neuman, RE and, Logan, MA., 1950). In summary hydroxyproline was hydrolyzed by heating the dried
scab with 6NHydrochloric acid followed by reaction with p-dimethyl amino benzaldehyde to get red coloration.
The red color thus formed was read on UV-Visible spectrophotometer (Elico SL 159) at 558 nm against reagent
blank. The amount of hydroxyproline in the scab was extrapolated from the calibration curve (Fig. S1) of standard
hydroxyproline (Merck).
1.5.4
Incision wound model
In this model six cm long paravertebral incisions were made through the full thickness of the skin on either side of
the vertebral column of anaesthetized rats. The wounds were closed with the interrupted sutures of one cm apart
using black surgical thread and curved needle no. 19. The sutures were pulled tight enough to secure good
adaption of the wound edges and the wounds were left undressed ( Lee, K.H., 1968; Seid MH et al., 1995). The
groups and the treatments given to the animals remained same as described in 4.5.2. The sutures were removed
on 8th post wounding day and the breaking strength of the newly formed skin was determines on day 10 using
tensiometer (Kokane DD et al, 2010.). The breaking strength is expressed as weights in gm required for breaking
open the healed incision wound.
1.6 Statistical Analysis
The data were expressed as mean ±SEM and the statistical significance was determined using an analysis of
variance (ANOVA) followed by Dunnett’s test. Values were considered significantly different at p<0.05.
References
Assimopoulou, A.N., Ganzera, M., Strupper, H., & Papageorgiou, V.P. (2007). Simultaneous determination of
monomeric and oligomeric alkanins and shikonins by high performance liquid chromatography diode array
detection-mass spectrometry. Biomedical Chromatography , 21 , 173-190.
Kokane, D.D., More, R. Y., Kale, M. B.,Gadgoli, C.H., Mehendale P.C., &Nehte, M.N. (2009). Evaluation of Wound
Healing Activity of Mimosa pudica Journal of Ethnopharmacology , 124, 311-315.
Lee, K.H., (1968) Studies on mechanism of action of salicylates: retardation of wound healing by aspirin. Journal of
Pharmaceutical Sciences, 57, 1042-1043.
Morton, JJ. & Malone, MH, (1972) Evaluation of vulnerary activity by an open procedure in rats. Archives
Internationales de Pharmacodynamie et de Therapie 196, 117-126.
Neuman, R.E. & Logan, M.A., ( 1950) The determination of hydroxyproline. The Journal of Biological Chemistry,
299-306.
Spyros, A., Papageorgiou, V.P., & Assimopoulou, A.N. (2005) Structure determination of oligomeric alkanin and
shikonin derivatives. Biomedical Chromatography, 19, 498-505.
Absorbance at 558
nm
1.5
1
y = 0.0126 + 0.0532
r2 = 0.9983
0.5
0
0
20
40
60
80
100
120
Concentration of Hydroxy proline in ppm
Fig. S1: Standard curve for Hydroxyproline
Visible light
a
b
c
d
e
f
Fig. S2 HPTLC Chromatogram of Petroleum Ether Extract of Root Bark of O. echioides and OE1
Track
Description
a
OE1: Isolated Compound 2ul
b
OE1: Isolated Compound 4ul
c
OE1: Isolated Compound 6ul
d
OE1: Isolated Compound 8ul
e
OE1: Isolated Compound 10 ul
f
PEOE 6 ul
1. Concentration of standard OE1: 1000 ppm.
2. Concentration of pet. ether extract (PEOE) 2000 ppm.
120
100
% Epithelization
80
**
*
60
40
**
*
**
**
*
**
*
**
*
**
*
**
*
**
*
**
*
**
*
CT (Control )
*
STD
**
*
F1 (PEOE 0.5% w/w)
F2 (PEOE 1% w/w)
F3(PEOE 2% w/w)
20
F4(OE 1-0.25% w/w)
0
Day4
day6
Day 8
Day 10
Day 12
Day 14
Day 16
Fig. S3 Effect of Topical applications of petroleum ether (60-800C) extract of Root bark of O echioides
(PEOE) and OE1 on % Epithelization of excision wound in rats
*P<0.05; **P< 0.01; ***P<0.001 ; No. of animals in each group = 6
Days of Epithelization
Period of Epithelization
30
26
25
***
18
20
19
19
19
***
15
11
10
5
0
CT (Control )
STD
F1 (PEOE F2 (PEOE 1% F3(PEOE 2% F4(OE 10.5% w/w)
w/w)
w/w)
0.25% w/w)
Fig. S4 Effect of Topical applications of petroleum ether (60-800C) extract of Root bark of O echioides
(PEOE) and OE1 on Period of Epithelization of excision wound in rats
***P<0.001 ; No. of animals in each group = 6