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Volume 15, Issue 3 (July 2021)
IJT 2021, 15(3): 135-142 |
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Karuppiah Pillai M. Evaluation of the Extracts From Rhizomes of Polygonum bistorta for the Median Lethal Dosages in Swiss Albino Mice. IJT 2021; 15 (3) :135-142
URL: http://ijt.arakmu.ac.ir/article-1-877-en.html
URL: http://ijt.arakmu.ac.ir/article-1-877-en.html
Department of Chemistry, Faculty of Science, National University of Singapore, Singapore, Republic of Singapore. , kmharan@rediffmail.com
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Introduction
Known by other names, such as Bistort and Snakeroot, Polygonum bistorta (P. bistorta) belongs to the Polygonaceae family [1-3]. This plant has been used in traditional Indian, Chinese and Japanese medicine as a remedy for a number of conditions, such as measles, jaundice, smallpox, pimples, cholera, dysentery, worms, insect stings and snakebites [3]. It has also been used as an astringent [1, 2] and applied topically to wounds to stop bleeding. The roots and leaves of P. bistorta have been used as food ingredients in Europe and America [4, 5]. The anticancer [2], anti-inflammatory [6], antibacterial [7, 8], antifungal [9] and antioxidant activities [9, 10] of the P. bistorta extracts have previously been reported. However, to the best of our knowledge, the median lethal dosage (LD50) of P. bistorta has not been reported previously. The objective of this study was to determine the LD50 of the crude extract and the hexane and chloroform fractions of P. bistorta in Swiss albino mice. The results are reported in this article. This is the first report on LD50 values of P. bistorta extracts.
Materials and Methods
Plant materials: Twelve grams of dried P. bistorta was obtained from a local market in Singapore. A voucher specimen viz. KMano PB2003 was issued at the Herbarium, Department of Biological Sciences, National University of Singapore (NUS).
Extraction procedures: The powdered plant materials were macerated with chloroform. Approximately, 250g of the brown residue from the chloroform crude extract was obtained after the removal of the solvent under vacuum. Approximately, 50g of the crude extract was kept separately for the MTT cytotoxicity assay and lethal dosage (LD50) analyses. The remaining crude extract (200g) was dissolved in water/methanol (95:5 v/v) solvent mixture. The mixture was subjected to solvent-solvent partition, first with hexane and then with chloroform. Approximately, 117 and 82 grams of the brown residues of hexane and chloroform fractions, respectively, were obtained after the removal of the solvents under vacuum. For the remaining methanol/water fraction, much of the solvent was removed under vacuum followed by freeze-drying to eliminate the solvent’s remnants. One gram of the yellowish-brown residue of methanol/water fraction was obtained. The crude extract, and the hexane and chloroform fractions were evaluated for their LD50 values. However, we did not evaluate the LD50 of the methanol/water fraction due its low quantity.
Solvents: The analytical reagent grades of hexane, chloroform and methanol were obtained from Sigma-Aldrich (St. Louise, MS, USA). The analytical grade of Dimethyl Sulphoxide (DMSO) was purchased from Merck Chemicals GmbH (Darmstadt, Germany). Deionized water was used for solvent-solvent partition.
Animal grouping & experimental procedures: In this study, 16 Swiss albino mice of both sexes, each weighing about 30g, were used and divided into four groups of four mice each. They were kept in the laboratory to get accustomed to the experimental environment. The crude extract, and the hexane and chloroform fractions, respectively, were given to the first three groups of rats with the fourth group being the controls. Solutions of 150 and 200mg of the crude extract, and the hexane and chloroform fractions per kg body weight of the mice were prepared separately in 10mL of 100% DMSO. These solutions were injected Intraperitoneally (IP) into the mice in groups 1, 2 and 3. The mice in group 4 (controls) received only 10 mL of 100% DMSO. The mice in all groups received normal diet. The general behaviors of the mice were observed for two weeks as per previously described procedures [11, 12]. On day 15, the mice were sacrificed by cervical dislocation and were used for further analyses.
Determination of the median Lethal Dosage (LD50): The dose that caused the death of 50% of the experimental mice was defined as LD50. Substances with LD50 values ranging between 1 and 5,000mg/kg of the mice’s body weight was considered as practically important. The LD50 values <1 and 5,000mg/kg were regarded as very toxic and practically not important in this study [13, 14]. The LD50 of the crude extract, and the hexane and chloroform fractions of P. bistorta were determined based on the previously described procedures [13-15]. Finney’s method of transformation of percentage mortalities to probits and the plot of probits (in ordinate) versus log doses (in abscissa) were used to determine LD50 [16-18]. Probit is the quantile function associated with the standard normal distribution.
Extraction procedures: The powdered plant materials were macerated with chloroform. Approximately, 250g of the brown residue from the chloroform crude extract was obtained after the removal of the solvent under vacuum. Approximately, 50g of the crude extract was kept separately for the MTT cytotoxicity assay and lethal dosage (LD50) analyses. The remaining crude extract (200g) was dissolved in water/methanol (95:5 v/v) solvent mixture. The mixture was subjected to solvent-solvent partition, first with hexane and then with chloroform. Approximately, 117 and 82 grams of the brown residues of hexane and chloroform fractions, respectively, were obtained after the removal of the solvents under vacuum. For the remaining methanol/water fraction, much of the solvent was removed under vacuum followed by freeze-drying to eliminate the solvent’s remnants. One gram of the yellowish-brown residue of methanol/water fraction was obtained. The crude extract, and the hexane and chloroform fractions were evaluated for their LD50 values. However, we did not evaluate the LD50 of the methanol/water fraction due its low quantity.
Solvents: The analytical reagent grades of hexane, chloroform and methanol were obtained from Sigma-Aldrich (St. Louise, MS, USA). The analytical grade of Dimethyl Sulphoxide (DMSO) was purchased from Merck Chemicals GmbH (Darmstadt, Germany). Deionized water was used for solvent-solvent partition.
Animal grouping & experimental procedures: In this study, 16 Swiss albino mice of both sexes, each weighing about 30g, were used and divided into four groups of four mice each. They were kept in the laboratory to get accustomed to the experimental environment. The crude extract, and the hexane and chloroform fractions, respectively, were given to the first three groups of rats with the fourth group being the controls. Solutions of 150 and 200mg of the crude extract, and the hexane and chloroform fractions per kg body weight of the mice were prepared separately in 10mL of 100% DMSO. These solutions were injected Intraperitoneally (IP) into the mice in groups 1, 2 and 3. The mice in group 4 (controls) received only 10 mL of 100% DMSO. The mice in all groups received normal diet. The general behaviors of the mice were observed for two weeks as per previously described procedures [11, 12]. On day 15, the mice were sacrificed by cervical dislocation and were used for further analyses.
Determination of the median Lethal Dosage (LD50): The dose that caused the death of 50% of the experimental mice was defined as LD50. Substances with LD50 values ranging between 1 and 5,000mg/kg of the mice’s body weight was considered as practically important. The LD50 values <1 and 5,000mg/kg were regarded as very toxic and practically not important in this study [13, 14]. The LD50 of the crude extract, and the hexane and chloroform fractions of P. bistorta were determined based on the previously described procedures [13-15]. Finney’s method of transformation of percentage mortalities to probits and the plot of probits (in ordinate) versus log doses (in abscissa) were used to determine LD50 [16-18]. Probit is the quantile function associated with the standard normal distribution.
Results
Animal behavior over time: The observed behavioral changes of the mice after IP injection of 150mg of the crude extracts, and the hexane and chloroform fractions over the 15 days of experiment are summarized in Table 1. The result indicated that the LD50 for the crude extract was ~150mg/kg, and for both the hexane and chloroform fractions was >150mg/kg (Table 1).
Since, the LD50 for both the hexane and chloroform fractions was >50 mg/kg, we repeated the experiment in order to establish the exact LD50 accurately. However, this time the mice in Groups 1, 2 and 3 received 200mg of each of the extracts per kg of the body weight. The results are presented in Table 2, indicating that the LD50 for the crude extract and the hexane and chloroform fractions were <200, >200 and ~200mg/kg, respectively. Therefore, the LD50 for the crude extract was established more realistically at ~150mg/kg, while the values for the hexane and chloroform fractions remained unchanged (Tables 1 & 2).
Determination of LD50: The injected and log doses, number of dead mice, percent mortalities for each of the doses, and the probits are summarised in Table 3.
The percent mortalities for each dose were transformed to probits based on Finney’s method [16-18] (Table 4).
The standard errors for the LD50 values were obtained using the Equation 1 [17, 18]:
1: SE of LD50 = (Log LD84 - LogLD16)/2N.
Crude extract: The plot of log doses vs probits of the crude extract of P. bistorta is presented in Figure 1. The LD50 value of the crude extract was calculated by extrapolation from the graph of the linear regression (Figure 1). The partial responses were obtained from the two experimental doses [15]. Also, the dose corresponding to probit 5 was the LD50 value of the crude extract (Figure 1). The probits of LD84 and LD16 of the crude extract were found to be 5.99 and 4.01, respectively, which were rounded-off to 6 and 4 (Table 2). Based on the graph, the log doses for the probits 6 and 4 were found to be 2.38 and 1.93, respectively, with the antilogs being 239.55 and 85.13, respectively. Substituting these values in approximate standard error equations resulted in a SE of 54.59. Therefore, the LD50 of the crude extract was 142.82±54.59 mg/kg based on the IP administration of the extract (Figure 1).
Chloroform extract: The plot of log doses versus probits for the chloroform fraction of P. bistorta is presented in Figure 2. The probits of LD84 and LD16 of the crude extract were 5.99 and 4.01, rounded-off to 6 and 4, respectively (Table 2). From the graph, the log doses for these were 2.50 and 2.10, respectively, and their antilogs were 315.14 and 127.15, respectively. Substituting these values for the approximate standard error equation resulted in a standard error of 66.47. Therefore, the LD50 of the chloroform extract was 200.17±66.47mg/kg when administered intraperitoneally (Figure 2).
Hexane extract: The hexane extract showed no mortality at either 150 or 200mg/kg. Supposedly, the hexane fraction may have an LD50 value >200 mg/kg in mice. For the same reason, it was impossible to calculate its exact LD50 from the graph since the plot of log doses versus probits requires at least partial responses [15]. Therefore, it was necessary to repeat the experiment with higher dosage of the hexane extract to determine the accurate LD50 value. However, we did not conduct further experiments since this in vivo study on mice required significantly larger quantity of the extract, which was not available. The methanol/water fraction was not investigated for its LD50 due to its low quantity available. We had to use large amounts of the crude extract, and the hexane and chloroform fractions for their MTT cytotoxic screening [1, 2] against murine and human cancer cell lines in addition to this in vivo study. The result of MTT cytotoxicity screening was already published [1, 2]. The cytotoxicity-guided fractionation of the hexane and chloroform fractions by chromatography resulted in several sub-fractions and the isolation of both known and new secondary metabolites [1, 2].
Since, the LD50 for both the hexane and chloroform fractions was >50 mg/kg, we repeated the experiment in order to establish the exact LD50 accurately. However, this time the mice in Groups 1, 2 and 3 received 200mg of each of the extracts per kg of the body weight. The results are presented in Table 2, indicating that the LD50 for the crude extract and the hexane and chloroform fractions were <200, >200 and ~200mg/kg, respectively. Therefore, the LD50 for the crude extract was established more realistically at ~150mg/kg, while the values for the hexane and chloroform fractions remained unchanged (Tables 1 & 2).
Determination of LD50: The injected and log doses, number of dead mice, percent mortalities for each of the doses, and the probits are summarised in Table 3.
The percent mortalities for each dose were transformed to probits based on Finney’s method [16-18] (Table 4).
The standard errors for the LD50 values were obtained using the Equation 1 [17, 18]:
1: SE of LD50 = (Log LD84 - LogLD16)/2N.
Crude extract: The plot of log doses vs probits of the crude extract of P. bistorta is presented in Figure 1. The LD50 value of the crude extract was calculated by extrapolation from the graph of the linear regression (Figure 1). The partial responses were obtained from the two experimental doses [15]. Also, the dose corresponding to probit 5 was the LD50 value of the crude extract (Figure 1). The probits of LD84 and LD16 of the crude extract were found to be 5.99 and 4.01, respectively, which were rounded-off to 6 and 4 (Table 2). Based on the graph, the log doses for the probits 6 and 4 were found to be 2.38 and 1.93, respectively, with the antilogs being 239.55 and 85.13, respectively. Substituting these values in approximate standard error equations resulted in a SE of 54.59. Therefore, the LD50 of the crude extract was 142.82±54.59 mg/kg based on the IP administration of the extract (Figure 1).
Chloroform extract: The plot of log doses versus probits for the chloroform fraction of P. bistorta is presented in Figure 2. The probits of LD84 and LD16 of the crude extract were 5.99 and 4.01, rounded-off to 6 and 4, respectively (Table 2). From the graph, the log doses for these were 2.50 and 2.10, respectively, and their antilogs were 315.14 and 127.15, respectively. Substituting these values for the approximate standard error equation resulted in a standard error of 66.47. Therefore, the LD50 of the chloroform extract was 200.17±66.47mg/kg when administered intraperitoneally (Figure 2).
Hexane extract: The hexane extract showed no mortality at either 150 or 200mg/kg. Supposedly, the hexane fraction may have an LD50 value >200 mg/kg in mice. For the same reason, it was impossible to calculate its exact LD50 from the graph since the plot of log doses versus probits requires at least partial responses [15]. Therefore, it was necessary to repeat the experiment with higher dosage of the hexane extract to determine the accurate LD50 value. However, we did not conduct further experiments since this in vivo study on mice required significantly larger quantity of the extract, which was not available. The methanol/water fraction was not investigated for its LD50 due to its low quantity available. We had to use large amounts of the crude extract, and the hexane and chloroform fractions for their MTT cytotoxic screening [1, 2] against murine and human cancer cell lines in addition to this in vivo study. The result of MTT cytotoxicity screening was already published [1, 2]. The cytotoxicity-guided fractionation of the hexane and chloroform fractions by chromatography resulted in several sub-fractions and the isolation of both known and new secondary metabolites [1, 2].
Discussion
In this study, the crude and the hexane and chloroform extracts of P. bistorta were evaluated for their LD50 values. Our study revealed that the crude extract might have contained all of the toxic ingredients. The successive extracts’ fractionation with hexane and chloroform might have distributed it’s the toxic ingredients. For the same reason, the crude extract showed an LD50 value of 142.82±54.59 mg/kg compared to the hexane and chloroform fractions. The hexane fraction might have contained mostly non-polar ingredients and the toxic effect might have been less than those of the extracts of the crude and chloroform fractions, showing a higher LD50 value >200mg/kg. On the other hand, the chloroform extract might have contained mostly the polar ingredients, hence the toxic effect being between those of the crude and hexane extracts (200.17±66.47mg/kg).
The pharmacological and biological activities of the various fractions of P. bistorta extracts have previously been reported. Its aqueous ethanolic extract has shown strong anti-inflammatory effect in experimental rats [6], with the active ingredients known as friedelinol and alnusenone (5-glutinen-3-1) [19]. The aqueous extract of P. bistorta has inhibited the mutagenicity of tryptophane pyrolysis product 1 (Trp-P-1) [20]. The cytotoxic activity of the extracts of the crude, hexane and chloroform fractions have been described previously [2]. Also, the antioxidant, antifungal and antibacterial activities of the extracts of P. bistorta have been reported previously [7-10]. Recently, the modulation of proteostasis by ROS-induced endoplasmic reticulum stress in human hepatoma cells by aqueous extract of P. bistorta has been reported [21]. The active ingredients of P. bistorta have been identified to be steroids, triterpenes, cycloartane-type triterpenes [1, 2, 22], phenolics [23, 24], tannins [8] and flavonoids [25].
The pharmacological and biological activities of the various fractions of P. bistorta extracts have previously been reported. Its aqueous ethanolic extract has shown strong anti-inflammatory effect in experimental rats [6], with the active ingredients known as friedelinol and alnusenone (5-glutinen-3-1) [19]. The aqueous extract of P. bistorta has inhibited the mutagenicity of tryptophane pyrolysis product 1 (Trp-P-1) [20]. The cytotoxic activity of the extracts of the crude, hexane and chloroform fractions have been described previously [2]. Also, the antioxidant, antifungal and antibacterial activities of the extracts of P. bistorta have been reported previously [7-10]. Recently, the modulation of proteostasis by ROS-induced endoplasmic reticulum stress in human hepatoma cells by aqueous extract of P. bistorta has been reported [21]. The active ingredients of P. bistorta have been identified to be steroids, triterpenes, cycloartane-type triterpenes [1, 2, 22], phenolics [23, 24], tannins [8] and flavonoids [25].
Conclusions
This study evaluated the extracts of the crude, hexane and chloroform fractions of P. bistorta for their LD50 values in Swiss albino mice. The LD50 were found to be 142.82, >200 and 200.17mg/ kg of the mice body weight, respectively. To the best of the authors’ knowledge, this is the first report of its kind on the LD50 values of P. bistorta plant in Swiss albino mice.
Ethical Considerations
Compliance with ethical guidelines
This study was approved by the National University of Singapore (NUS).
Funding
This study was extracted from the the PhD. dissertation of the first author in the Department of Chemistry, Faculty of Science, National University of Singapore.
Conflict of interest
The authors declared no conflict of interest.
Acknowledgements
The author thanks the National University of Singapore for the meritorious PhD. Fellowship. MK Pillai also acknowledges the assistance provided by Ms. Annie Hsu at Traditional Medicines and Natural Products Research Laboratory, Department of Pharmacology, NUS.
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