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American Journal of Ethnomedicine
ABSTRACT
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American Journal of Ethnomedicine ________________________________________ ISSN: 2348-9502
temperature (22 1C) and humidity for at each 100 L of plasma sample, 100 L of
least one week before the beginning of standard drug concentration and 100 L
experiment. They had free access to food internal standard (zidovudine) was added.
and water. Approval of Institutional Animal Finally 100 L acetonitrile was added and
Ethical Committee (IAEC Reg. No: vortexed for 1 min and centrifuged at 10000
1047/ac/07/CPCSEA, dated 24-04-2007) g for 3 min. The supernatant was transferred
was taken to perform the experiments. to clean, similarly labeled tube and was
subsequently recentrifuge at 10000 g for 2
Estimation of ritonavir by sensitive RP min and then evaporate the acetonitrile from
HPLC method these tubes and add the mobile phase. These
samples were stored at -20C until use. The
HPLC description resultant solution was injected into HPLC.
A Waters HPLC system is used in Peak area ratios were calculated using the
the study consisted of a pump (Model formula: Peak area ratio = Peak area of
Waters 515 HPLC pump) operating at 1 drug/Peak area of internal standard.
mL/min, a syringe loading sample injector
of 20 L capacity, a C18 reverse phase Construction of calibration curve
column of 250 4.6 mm dimension and 5 The calibration curve was
particle size and a dual wavelength UV- constructed using peak area ratios of drug to
Visible detector (UV-1800). The data internal standard vs nominal concentration.
analysis was done by Wufeng-chrom The slope of plot determined by the least
workstation (Shanghai Wufeng Scientific square regression analysis was used to
Instruments Co. Ltd Shangai). calculate the ritonavir concentrations in the
unknown sample. A linear calibration curve
Chromatographic conditions in the range of 0.1 to 7 g was established
The mobile phase consisted of (r2 = 0.9993). Retention times of zidovudine
acetonitrile: water (60: 40 % v/v) was and ritonavir were 2.65 and 8.68 min,
pumped at a flow of 1 mL/min. The mobile respectively.
phase was filtered through 0.4 m
membrane filter and degassed before use. Experimental design of pharmacokinetic and
The effluent was monitored at 240 nm. The toxicological studies in normal healthy rats
total run time of the method was set at 15 Wistar rats of either sex were
min. randomly distributed into four groups of six
animals in each group; they are housed in
Preparation of calibration curve of ritonavir well ventilated aluminum cages and
for in vivo samples maintained on uniform diet and temperature
Stock solutions of 1000 g/mL of with 12 h light and dark cycle. Before
ritonavir and zidovudine (internal standard) experiment all animals fasted for Overnight
were prepared by dissolving in required and water ad libitum, water was withdrawn
quantity of acetonitrile. From the stock during experiment. After collection of initial
solution further dilutions of 0.1, 0.3, 0.5, blood samples, drugs were administered
0.7, 1, 3, 5, 7 g/mL of ritonavir were orally. The animals were divided into
prepared by using acetonitrile. 100 g/mL of various groups in the following way:
zidovudine was prepared to be used as an Group I: Control (0.2 mL of 0.5% CMC
internal standard. A standard graph was sodium).
prepared by adding a known concentration Group II: Receive ritonavir (30 mg/kg) -
of ritonavir to drug free plasma. Briefly, to Single dose.
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American Journal of Ethnomedicine ________________________________________ ISSN: 2348-9502
Group III: Receive ritonavir (30 mg/kg) + For Standard AS = A2S - A1S.
ashwagandha (300 mg/kg)- Single dose. For Test AT = A2T - A1T.
Group IV: Administer ashwagandha (300 Calculate the Creatinine in mg/dl:
mg/kg) for 9 days. Creatinine in mg/dl = ( AT/ AS) x 2.0
On 9th day administer (10 mg/kg) of
ritonavir. Data analysis
Blood (0.5 mL) for analysis was The data obtained was pooled for
collected from orbital sinuses using each group. Data were analyzed by one way
heparinized capillaries into a micro ANOVA followed by Dunnetts test for
centrifugation tubes contain anticoagulant at comparison of all other columns with
1, 2, 4, 8 and 12 h after treatment. Plasma control and at a significance level of p <
was analyzed separated by centrifugation 0.05. The data is presented as mean SD.
and stored at -20C until further analysis.
These samples are used to analyze for Estimation of complete blood picture (CBP):
pharmacokinetic and toxicological i.e Automated blood count
SGOT-SGPT, Complete Blood Picture The blood is well mixed (though not
(CBP), Serum Creatinine Clearance test. shaken) and placed on a rack in the analyzer
Ritonavir levels were estimated by a (Cobus u 411). The cell counting component
sensitive RP-HPLC method. counts the numbers and types of different
cells within the blood.
Estimation of SGOT-SGPT levels by Blood (0.1 mL) is aspirated through
modified IFCC-UV kinetic method narrow tubing which contains sensors that
Enzyme reagent (1 mL) was added to count the number of cells going through it,
0.1 mL of plasma at room temperature. It and can identify the type of cell by flow
was mixed well and absorption was cytometry. The two main sensors used are
measured against blank at 340 nm. The light detectors, and electrical impedance.
initial absorbance A0 was measured after One way the instrument can tell what type of
exactly 1 min. A1, A2 and A3 were blood cell is present is by size. Other
measured after every 30 s for 1 min 30 s. instruments measure different characteristics
The average change in absorbance of the cells to categorize them.
per minute ( A/min) was calculated by the Automated hematology analyzers
formula: also measure the amount of hemoglobin in
IU/L = A/min X 1746 X tf the blood and within each red blood cell.
Temperature conversion factor (tf) =1.
RESULTS AND DISCUSSION
Estimation of serum creatinine levels by
modified JaffeS kinetic method Ritonavir is well absorbed from the
Enzyme reagent (1 mL) was added to gastrointestinal tract following oral dosing.
0.1 mL of plasma at room temperature. It Peak plasma concentrations are achieved
was mixed well and absorption was with in 3-5 h. It is 98-99% bound to plasma
measured against blank at 520 nm. The proteins. It is extensively metabolized in
initial absorbance A1 for the Standard and liver, almost exclusively by the cytochrome
Test were measured after exactly 30 s. P450 isoenzyme CYP3A4. Toxicity studies
Another absorbance A2 of the Standard and in animals identified major target organs as
Test were measured exactly 60 s later. A the liver, retina, thyroid gland and kidney.
for both the Standard and Test were Hepatic changes involved hepatocellular,
calculated. biliary and phagocytic elements and were
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American Journal of Ethnomedicine ________________________________________ ISSN: 2348-9502
accompanied by increases in hepatic treated group (Table 3). The elevated levels
enzymes18. could be because of the induction caused by
the CYP that ultimately lead to toxicity
Pharmacokinetics of ritonavir after the oral which was controlled by ashwagandha. In
administration of standardized herbal extract contrary there is significant elevation in the
of ashwagandha serum creatinine levels in all the groups
In the present study it is quite treated (Table 3).
evident that single and multiple doses of Ashwagandha is a very popular
ashwagandha decreased the plasma Indian traditional medicine and is used as a
concentration of ritonavir. There is general tonic. It has been found to possess
significant decrease in the AUC and increase potent immunomodulatory actions. It is
in clearance. In the single dose studies the often sold on OTC even by druggists and
change in PK parameters could be because chemists as a health supplement. HIV
of the transient induction in intestinal P-gp patients are immuno-compromised and
and CYP 3A4. When ashwagandha was because ashwagandha is a general tonic with
given in repeated doses (9 days) there could immunomodulatory effects, it is possible
be induction of both the intestinal and that both are concomitantly used by such
hepatic CYP 3A4 subsequently increasing patients. Therefore, there is a chance for
the metabolism and clearance of ritonavir, herb-drug interaction in HIV patients with
decreasing the overall AUC by more than the use of ashwagandha and ritonavir.
50% on repeated administration of The above studies revealed that
ashwagandha (Table 1) showing a potential plasma concentrations of ritonavir decreased
metabolic interaction. The decrease in in the presence of ashwagandha below the
plasma concentration of ritonavir and AUC MEC. The ritonavir plasma concentrations
may have direct impact on the decreased significantly both in single and
pharmacodynamics in terms of the viral multiple doses groups of ashwagandha.
load. There are significant changes in the PK
parameters. In single dose groups of
Toxicity of ritonavir in the presence of ashwagandha - ritonavir AUC decreased
standardized herbal extract of ashwagandha with increase in clearance. Induction of
In the present experiments blood CYP3A4 by repeated administration of
profile, liver (SGPT, SGOT) and kidney ashwagandha could be the major reason for
(serum creatinine) function tests were the decrease in the AUC. Ashwagandha
studied to correlate the effect of the protected the deleterious effects caused by
ashwagandha-ritonavir combination on the ritonavir on the blood cells and liver, it
toxicity profiles. Ashwagandha have clearly could not protect those effects on kidney.
shown the beneficial effect in the CBP,
where there is significant improvement in CONCLUSION
blood cell count and lymphocytes in
particular (Table 2). Studies clearly indicate The studies clearly indicate a
the beneficial effect of ashwagandha on the potential interaction (metabolic) between
deleterious effects caused by ritonavir on ashwagandha and ritonavir. The interaction
blood profile and most importantly could could lead to sub therapeutic levels of
help the ailing immunity of the HIV patients ritonavir and obvious increase in the viral
on ritonavir therapy. Ashwagandha also load and failure of the therapy. Hence the
improved the liver function compared to the present study recommends that while such
elevated levels of SGPT/SGOT in ritonavir herb drug combinations are used, dosage
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American Journal of Ethnomedicine ________________________________________ ISSN: 2348-9502
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American Journal of Ethnomedicine ________________________________________ ISSN: 2348-9502
Table 1. Comparison of pharmacokinetic parameters of ritonavir (30 mg/kg) and also following
pretreatment with ashwagandha (300 mg/kg) by oral administration in healthy rats (n = 6)
Ritonavir Ritonavir
+ +
Parameter Ritonavir
Ashwagandha Ashwagandha
(1stday) (9th day)
AUC (g/h) 52.67 3.37 34.74 1.68*** 22.74 1.61***
AUMC (g/ml/h) 61.08 2.16 55.71 1.92*** 60.5 1.57
CL_F (L/h) 83.12 2.42 124.84 4.42*** 163.81 5.64***
CL_F (L/h/kg) 576.49 19.68 853.25 15.78*** 1071.39 28.72***
Tmax (h) 1.06 0.03 1.06 0.03 1.04 0.028
Cmax (g/mL) 12.45 0.74 10.59 0.38*** 9.32 0.49***
MRT (h) 5.47 0.33 1.60 0.10** 1.20 0.09***
Table 2. Complete blood picture (CBP) (mean SD) changes after oral administration of
ritonavir and combination with ashwagandha in healthy rats (n = 6)
Ritonavir Ritonavir
+ +
Cells Control Ritonavir
Ashwagandha Ashwagandha
(1stday) (9th day)
Haemoglobin (g) 14.3 0.21 12.9 0.16** 13.55 0.327*** 14.21 0.40
R.B.C (m/cmm) 4.98 0.04 4.03 0.103** 4.2 0.33*** 4.98 0.04
Total count (TC) (m/cmm) 4008.33 66.45 3616.66 354.49* 3823.33 2.065 4166.66 163.29
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Table 3. Mean SD changes in SGOT, SGPT and serum creatinine levels after oral
administration of ritonavir and combination with ashwagandha in healthy rats (n = 6)
Serum creatinine levels
Group SGOT (U/L) SGPT (U/L)
(mg/dL)
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