Analytical, biological, pharmacokinetic and stability studies of piper sarmentosum roxb. extracts and selected studies of orthosiphon stamineus benth. extracts
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Date
2008
Authors
Hussain, Khalid
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Abstract
The study aimed to perform analytical and biological activity studies on root,
stem, leaf and fruit of Piper sarmentosum and leaves of Orthosiphom stamineus,
pharmacokinetic and stability studies on ethanol extract of fruit of Piper
sarmentosum. Powdered material of different parts of Piper sarmentosum was
extracted using ethanol and water while leaves were also extracted sequentially using
petroleum ether, chloroform and methanol. Leaves of Orthosiphom stamineus were
extracted with methanol and few prepared extracts were obtained from different
sources. The extracts of both the plants were analyzed qualitatively and
quantitatively using Fourier Transform Infrared (FTIR) and ultraviolet/visible
(UV Nis) spectroscopy, high performance thin layer chromatography (HPTLC) and
high performance liquid chromatography (HPLC). Aqueous and ethanol extracts of
different parts of Piper sarmentosum were investigated for in vitro antioxidant
activity and the extracts having good activity namely fruit and leaf ethanol extracts
were evaluated for in vivo antioxidant and hepatoprotective activity using CCL.
induced oxidative stress model. Sequential extracts of Piper sarmentosum leaf and
fractions of the methanol extract, aqueous extract of Orthosiphom stamineus and its
fractions were investigated for antiangiogenic and interaction studies. Ethanol extract
of the fruit of Piper sarmentosum was investigated for pharmacokinetic and
accelerated stability studies using pellitorine, sarmentine and sarmentosine as
markers.
Qualitative analysis indicated the presence of flavonoids and alkaloids in
ethanol extracts of different parts of Piper sarmentosum while flavonoids and
triterpenes in extracts of Orthosiphom stamineus. Quantitative analysis of different
extracts of Piper sarmentosum by HPTLC indicated varying amaunts of rutin
(0.0004-0.0109 mg/g) and naringenin (0.010-0.659 mg/g). The HPTLC analysis of
different extracts of Orthosiphon stamineus indicated varying amaunts of betulinic
acid (0.013-0.124 mg/g) and sinensitin (0.470-1.335 mg/g).
A new HPLC method was developed, validated and applied for the
simultaneous quantification of rutin and flavonone in Piper sarmentosum extracts
which indicated varying amaunt of rutin ranging 0.20-5.02 mg/g and flavonone 0.32-
15.32 mg/g. Another HPLC method was developed, validated and applied for the
simultaneous quantification of pellitorine, sarmentine and sarmentosine in Piper
sarmentosum extracts which indicated varying amaunts of pellitorine ranging 0.043-
6.820 mg/g, sarmentine 0.006-0.420 mg/g and sarmentosine 0.005-0.120 mg/g. A
previously used HPLC method for the quantification of betulinic acid was improved,
validated and applied to analyse different extracts of Orthosiphon stamineus which
exhibited varying amaunts ofbetulinic acid ranging 2.76-9.50 mg/g.
Ethanol extracts of the fruit and leaf of Piper sarmentosum exhibited good in
vitro antioxidant activities in DPPH model with ICso at 25.87 and 23.66 ~g/mL,
respectively. Both the extracts have also shown good activity in 13-carotene linoleate
model. The antioxidant activity of the extracts was found to be correlated with total
content ofpolyphenolics, flavonoids and amides (P < 0.05).
Acute oral toxicity studies of the fruit and leaf ethanol extracts of Piper
sarmentosum indicated median lethal dose (LD5o) above 2000 mg/kg in rats. The
extracts of both the parts were investigated for in vivo antioxidant and
hepatoprotective activities in two dose levels 250 and 500 mg/kg. The pretreated
groups exhibited significant preservation of antioxidant activity and liver function
markers as compared to negative control (CC4) group (P < 0.05).
Chloroform extract of the leaf of Piper sarmentosum and n-hexane fraction of
aqueous extract of Orthosiphon stamineus exhibited 100 and 80% antiangiogenic
activity with IC50 at 45 f.lg/mL and IC50 at 45 f.lg/mL, respectively. Amides isolated
from the fruit of Piper sarmentosum, pellitorine, sarmentine and sarmentosine,
exhibited 30 % antiangiogenic activity while the isolated compounds from the nhexane
fraction of Orthosiphon stamineus, betulinic, oleanolic and ursolic acids,
exhibited 100% antiangiogenic activity. In cytotoxicity studies, the chloroform
extract and the n-hexane fraction have shown-- IC50 at 76.24 and 80 f.lg/mL,
respectively, which are higher as compared to ICso of antiangiogenesis.
Chloroform and ethyl acetate fractions of the leaf methanol extract of Piper
sarmentosum have shown antimycobacterial activity with minimum inhibitory
concentration (MIC) 3.12 f.lg/mL while n-hexane fraction of aqueous extract of
Orthosiphon stamineus has shown the activity MIC 3.12 f.lg/mL. In interaction
studies, the extracts and the fraction of both the plants have not shown any
interaction with isoniazid because fractional inhibitory concentration index (FICI)
was found to be > 0.5.
A new HPLC method was developed, validated and applied for the
simultaneous determination ofpellitorine, sarmentine and sarmentosine from plasma,
urine, fecal matter and tissues. In pharmacokinetic studies of the fruit ethanol extract
of Piper sarmentosum in rats, pellitorine and sarmentine showed good oral
bioavailability while sarmentosine was not absorbed orally and excreted unchanged
in feces. Pellitorine and sarmentine exhibited different tissue affinities and were
excreted in urine as metabolites.
In accelerated stability studies, estimated shelf life (19o) of pellitorine,
sarmentine and sarmentosine was approximately 16 months at 25 °C. The markers
followed the zero order reaction and their stability was found to be decreasing at high
temperature and relative humidity (RH).
The results of the study indicates that the developed HPLC methods are
simple, easy to perform and can be applied for standardization. Extracts of Piper
sarmentosum have promising antioxidant and hepatoprotective activity. Chloroform
extracts of Piper sarmentosum and n-hexane fraction of aqueous extract of
Orthosiphon stamineus have promising antiangiogenic activity. The extracts of both
the plants have no interaction with isoniazid.
Description
Keywords
Pharmacokinetic , Piper sarmentosum roxb. , Orthosiphon stamineus benth.