Palmatine is a quaternary protoberberine alkaloid. It is typically yellow in color and reported as the most important pharmacological active constituents of a number of plants, such as
The purpose of the present study was to optimize the extraction conditions for the isolation of palmatine from
The stems of
Stems of
Response surface analysis was performed to estimate the effects of independent variables on the response within the range of investigation. RSM with the central composite design was used to analyze the experimental data with 3 independent variables (
Independent variables and their levels used in the response surface design.
Serial number | Independent variables | Symbols | Factor level | ||
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Low (−1) | Middle (0) | High (+1) | |||
1 | Extraction temperature (°C) |
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30 | 40 | 50 |
2 | Extraction time (hours) |
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16 | 32 | 48 |
3 | Extraction cycles (cycle) |
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4 | 12 | 20 |
Table
Central composite design by RSM program for optimization of extraction conditions with experimental and their corresponding predicted yields.
Runs | Extraction temperature |
Extraction time |
Extraction cycles |
Experimental yield (%) | Predicted yield (%) |
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1 | 40 (0) | 32 (0) | 12 (0) | 13.29 | 11.51 |
2 | 50 ( |
16 (−1) | 20 ( |
8.16 | 8.86 |
3 | 40 (0) | 32 (0) | 12 (0) | 10.26 | 11.51 |
4 | 30 (−1) | 48 ( |
20 ( |
11.32 | 11.09 |
5 | 23.18 (−1.68) | 32 (0) | 12 (0) | 12.89 | 13.09 |
6 | 56.82 (1.68) | 32 (0) | 12 (0) | 9.67 | 9.94 |
7 | 40 (0) | 5.09 (−1.68) | 12 (0) | 11.93 | 11.51 |
8 | 40 (0) | 32 (0) | −1.45 (−1.68) | 11.36 | 11.91 |
9 | 50 ( |
48 ( |
4 (−1) | 12.19 | 11.82 |
10 | 40 (0) | 32 (0) | 25.45 (1.68) | 10.83 | 11.12 |
11 | 30 (−1) | 48 ( |
4 (−1) | 11.97 | 11.20 |
12 | 40 (0) | 32 (0) | 12 (0) | 11.31 | 11.51 |
13 | 30 (−1) | 16 (−1) | 04 (−1) | 13.67 | 14.29 |
14 | 50 ( |
16 (−1) | 04 (−1) | 9.53 | 9.68 |
15 | 40 (0) | 58.91 (1.68) | 12 (0) | 9.67 | 11.52 |
16 | 40 (0) | 32 (0) | 12 (0) | 12.73 | 11.51 |
17 | 50 ( |
48 ( |
20 ( |
12.65 | 11.96 |
18 | 40 (0) | 32 (0) | 12 (0) | 12.42 | 11.51 |
19 | 30 (−1) | 16 (−1) | 20 ( |
12.92 | 13.21 |
Methanolic extract of stem was partitioned to CHCl3 and aqueous extract. The CHCl3 solution was dried and evaporated up to a brownish viscous residue (8 gm). The residue was placed on a silica gel column and eluted with CHCl3 and gradually enriched with methanol to afford 5 fractions. Fraction 3 eluted with CHCl3-MeOH (10 : 1) was repeated and subjected to silica gel column chromatography to give single compound (2.8 gm). Isolated compound was subjected to ultra-violet, infrared, gas chromatography mass spectrometry, and nuclear magnetic resonance spectroscopy. Spectroscopic analysis revealed the presence of a high content of the alkaloid palmatine. This material was further purified by recrystallization with methanol to yield palmatine (99% purity) [
7,12-Dimethylbenz(a)anthracene (DMBA), croton oil, NADH, glutathione reduced (GSH), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), and 2-thiobarbituric acid (TBA) were obtained from Sigma-Aldrich, USA. All other chemicals were commercially available and analytical grade.
Swiss albino mice were selected at random from animal house of the Pinnacle Biomedical Research Institute (PBRI), Bhopal. Animals were housed in polypropylene cages with sterile husk and provided standard pellet (Golden feeds, New Delhi) and water
According to Organisation for Economic Co-operation and Development (OECD) 423 guideline, the acute toxicity was performed on mice using different doses of palmatine (100, 200, 400, 600, 800, and 1000 mg/kg) orally on five mice. The mice were observed continuously for the first 2 hours and then occasionally for 24 hours and daily thereafter for 30 days for any signs of morbidity, mortality, peripheral blood changes, and behavioral toxicity. No mice were found to be dead during toxicity studies. So, we have selected the 1/5th and 1/10th of maximum exposed dose.
Five groups (five animals per group) of Swiss albino mice of either sex were used for the study. Animals were dorsally shaved with hair clipper.
Mice were observed each week for incidence of skin tumors and their sizes, body weight and average latency period till 16 weeks [
At the end of the experiment, animals of all the groups were sacrificed by cervical dislocation. The animals were immediately dissected to remove their skins which were washed in ice-cold saline (0.9% NaCl) and the extraneous material was removed. It was then weighed and blotted dry. The skin tissue homogenate was prepared in 0.15 M Tris-KCl (pH 7.4) and centrifuged at 12000 rpm for 15 minutes.
For biochemical estimation, postmitochondrial supernatant was used on the same day that animals were killed. The level of lipid peroxidase (LPO), glutathione (GSH), superoxide dismutase (SOD), and catalases was estimated by using different methods [
The blood was obtained from all animals by puncturing retro-orbital plexus. At room temperature, the blood samples were allowed to clot for 45 min. Serum was separated by centrifugation at 2500 rpm at 30°C for 15 min and utilized for the determination of various biochemical parameters. The levels of serum glutamate oxalate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), and serum bilirubin were estimated by using the span diagnostic kit.
Alkaline single cell gel electrophoresis (SCGE) was performed as a three-layer procedure [
At the end of the experiment, animals were sacrificed, and fresh portions of the skin from each animals were cut rapidly, fixed in neutral buffered formalin (10%), and then dehydrated using different grades of ethanol (70%, 80%, 90%, 95%, and 100%). Dehydration was followed by clearing the samples in two changes of xylene. The samples were then impregnated with two changes of molten paraffin wax, embedded, and blocked out. The tissue sections (4-5
The total protein content in skin tissue extracts was estimated by the Bradford method using bovine serum albumin as standard [
Design-Expert (Version 8.0) software was used to analyze the experimental data. Values are recorded as mean ± SD. The data obtained from different groups was analyzed by ANOVA. The values
The effect of extraction temperature, time, and cycles on extraction yield was estimated. According to the above extraction conditions, the yield of
The plot of the predicted values versus experimental value of the yield of
Analysis of variance for fitted 2FI polynomial model.
Source | Sum of square | Degree of freedom | Mean square |
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Model | 26.83 | 6 | 4.47 | 4.00 | 0.019 significant |
Residual | 13.40 | 12 | 1.12 | ||
Lack of fit | 7.52 | 8 | 0.94 | 0.64 | 0.726 not significant |
Pure error | 5.88 | 4 | 1.47 | ||
Cor total | 40.23 | 18 |
Correlation between predicted and experimental values of the yield of
Contour plots showing the effect of extraction temperature, extraction time, and extraction cycle on the yield of
The contour plots based on derived equation represent the relationship between the response and the experimental levels of each factor, by which the optimum condition for the maximum yield could be presumed. Different contour plots can reflect the strength of the interaction effects. According to Figure
A gradual decrease in body weight was observed in all animals of the different groups. Animals of Groups 4-5 gave a continuous treatment of palmatine orally as mentioned above along with the repeated application of croton oil and showed a significant reduction in the cumulative number of papillomas and tumor size (Table
Chemopreventive effect of palmatine against DMBA and croton oil induced skin carcinogenesis in mice.
Treatment | Final body weight (g) | Number of papilloma | Tumor size | Average latency perioda |
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Group 1 |
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Group 3 |
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Group 4 |
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Group 5 |
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Values with * superscripts were significant (
Flavonoid-induced reduction of tumor in Swiss albino mice. (a) Group 2 (water + DMBA + croton oil), (b) Group 4 (palmatine 100 mg/kg + DMBA + croton oil), (c) Group 5 (palmatine 200 mg/kg + DMBA + croton oil).
A significant increase in GSH, SOD, and catalase was noted in the skin of palmatine administered animals (Groups 4 and 5) than in the control group animals (Group 2) (Table
Inhibition of dimethylbenz(a)anthracene (DMBA)/croton oil induced skin carcinogenesis in Swiss albino mice by palmatine treatment.
Treatment | GSH |
SOD |
Catalase U/mg protein | LPO nmole/mg protein |
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Group 1 |
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Group 2 |
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Group 3 |
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Group 4 |
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Group 5 |
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Values with * superscripts were significant (
A significant decrease in enzyme serum glutamate oxalate transaminase and serum glutamate pyruvate transaminase, alkaline phosphatase, and bilirubin level was noted in the serum of palmatine administered animals (Groups 4 and 5) than the control group animals (Group 2) (Table
The effect of palmatine on serum enzyme and bilirubin levels in mice.
Treatment | SGOT | SGPT | SALP | Bilirubin mg/dL |
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IU/L | IU/L | IU/L | ||
Group 1 |
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Group 2 |
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Group 3 |
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Group 4 |
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Group 5 |
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Values with * superscripts were significant (
In general, the normal dermal layer consists of two layers in which one has loose connective tissue and another has dense connective tissue called the papillary and reticular layers, respectively. It was observed that no alterations occurred in the structure of Group 1 (water treated animal) and Group 3 (only palmatine treated animal) mice skin (Figures
Light microphotographs of cross-sections of mouse skin. (a) Group 1: mouse received normal water demonstrating normal histological architecture,H&E, 400X. (b) Group 2: mouse topical exposure to DMBA + croton oil demonstrating damage
The DNA damage was measured as % tail DNA in the control (Groups 1-2) as well as exposed Groups 3–5. The animal exposed to DMBA and croton oil exhibited significantly (
DNA damage in the lymphocytes after different exposures (a) % Tail DNA. (b) Group 1, (c) Group 2, (d) Group 3, and (e) Group 4. Each value represents the mean ± S.E. of three experiments.
The suitability of the model equation for predicting the optimum response values was evaluated using the optimal conditions. When the contour plots are oval, it means that the interaction of two independent variables is significant. In contrast, the round contour plots are considered not significant [
Human epidemiological data indicate that regular use of certain medicinal plants suppresses carcinogenesis in various organs [
Lipid peroxidation is a free radical chain reaction and is known to cause two main steps of carcinogenesis, that is, initiation and propagation. It is a highly destructive process. During the carcinogenic process, lipid peroxidation is increased and more complex and reactive compounds such as malondialdehyde (MDA) and 4-hydroxynonenal were formed. These products of lipid peroxidation were observed to be mutagenic and carcinogenic [
Glutathione (GSH) is a tripeptide nonenzymatic antioxidant with a single cysteine residue and constitutes an important pathway of the antioxidant and detoxification defense. GSH is essential for protection of the cells against reactive oxygen species and free radicals produced even in normal metabolism [
DMBA treatments induce lipid peroxidation and reactive oxygen species in the affected area of the skin and ultimately lead to carcinogenesis. This oxidative stress was easily observed in the control group as level of LPO was higher and the level of catalase, SOD, and GSH was lower. The beneficial action of palmatine is probably due to its ability to stimulate the antioxidant enzymes in the cells. This increase in enzyme activity effectively reduced the generation of ROS and LPO in the skin and thus might reduce the incidences of skin papillomas on the treated areas has reported that extensive DNA damage triggers apoptosis [
In conclusion, our results indicate that the extraction condition for
The authors declare that they have no conflicts of interests regarding this work.
The authors are grateful to Dr. Gajendra Dixit, Dean R & C, MANIT, Bhopal, India (Grant no. Dean R&C/2010/159), and Manvendra Karchuli, Research Officer, PBRI, Bhopal, India that support this research and that provided facilities to achieve the desired goals of the study.