|Year : 2021 | Volume
| Issue : 3 | Page : 291-297
Standardization of Leonurus sibiricus L. aerial part and capillary electrophoresis quantitative analysis of its leonurine content
Maneewan Suwatronnakorn1, Somchai Issaravanich1, Chanida Palanuvej1, Nijsiri Ruangrungsi2
1 Department of Public Health Sciences Program, College of Public Health Sciences, Chulalongkorn University, Bangkok, Thailand
2 Department of Public Health Sciences Program, College of Public Health Sciences, Chulalongkorn University, Bangkok; Department of Pharmacognosy, College of Pharmacy, Rangsit University, Pathum Thani, Thailand
|Date of Submission||08-Mar-2021|
|Date of Decision||26-Apr-2021|
|Date of Acceptance||18-Jun-2021|
|Date of Web Publication||16-Jul-2021|
Assoc. Prof. Dr. Nijsiri Ruangrungsi
College of Public Health Sciences, Chulalongkorn University, Bangkok
Source of Support: None, Conflict of Interest: None
The quality parameters of Leonurus sibiricus L. aerial part crude drugs were evaluated. Fifteen crude drugs were collected from various locations throughout Thailand. The transverse section of the stem of L. sibiricus showed quadrangular character highlighted the ribs with angular collenchyma. The epidermis was uniseriate with abundant glandular trichomes distribution. Prismatic calcium oxalate prisms were found in the stem medullary parenchyma. The histological character of crude drug powder showed bordered pitted vessel, fragment of fiber, glandular trichome, prism crystal, spiral vessel, starch granule, and stomata. The loss on drying, total ash, acid-insoluble ash, and moisture contents should be not more than 8.18, 15.28, 4.04, and 8.91 g/100 g dry weight, whereas ethanol and water-soluble extractive values should be not less than 7.67, and 17.21 g/100 g of dry weight, respectively. Leonurine in the crude drugs were analyzed by capillary electrophoresis (CE) with photodiode array detector. The ethanolic extraction performed by Soxhlet apparatus yielded 18.86 ± 4.09 g/100 g dry weight. The electropherogram detected at 277 nm showed the migration time of leonurine at 6.2 min. The developed CE was found to be valid for leonurine quantification in L. sibiricus ethanolic extract. The contents of leonurine in 15 crude drugs ranged from 0.79 to 4.23 mg/g with the average of 2.38 ± 1.10 mg/g dry weight. This study established the pharmacognostic specification of L. sibiricus crude drug in Thailand with special reference to a bioactive compound, leonurine. CE was beneficial technique for the analysis of leonurine in L. sibiricus aerial parts.
Keywords: Capillary electrophoresis, leonurine, Leonurus sibiricus L., standardization
|How to cite this article:|
Suwatronnakorn M, Issaravanich S, Palanuvej C, Ruangrungsi N. Standardization of Leonurus sibiricus L. aerial part and capillary electrophoresis quantitative analysis of its leonurine content. J Adv Pharm Technol Res 2021;12:291-7
|How to cite this URL:|
Suwatronnakorn M, Issaravanich S, Palanuvej C, Ruangrungsi N. Standardization of Leonurus sibiricus L. aerial part and capillary electrophoresis quantitative analysis of its leonurine content. J Adv Pharm Technol Res [serial online] 2021 [cited 2022 Aug 15];12:291-7. Available from: https://www.japtr.org/text.asp?2021/12/3/291/321512
| Introduction|| |
Leonurus sibiricus L. or Siberian motherwort, locally known in Thailand as “Khanchaa thet” or “Saa saa, Saa nam,” is an herbaceous plant belonging to the Labiatae family. It is an annual or biennial herb with stems erect, three lobes pinnately divided leaves, lobules of leaves 1–3 mm wide, flower sessile, corolla white or reddish to purple-red , calyx densely pilose, especially at the middle. L. sibiricus is confined to stony or sandy grasslands and Pinus forests in its original native distribution (Russia, Mongolia, and China). L. sibiricus has been used in traditional Thai herbal medicine, especially in women remedies for menstrual induction, preventing postpartum hemorrhage and improving blood circulation.,, The pharmacological study of L. sibiricus showed that the ethanolic extract of leaves and stems had promising activity on uterine contraction in Wistar rats. In addition, L. sibiricus is one of the main ingredients in Prapchompoothaweep remedy, which is listed in Thailand National List of Essential Medicines (NLEM), for traditional treatment of common cold and allergic rhinitis symptoms. The clinical trials of this remedy on allergic rhinitis treatment revealed significant improvement of the symptoms and better quality of life related to rhinoconjunctivitis., Drugs listed in NLEM must be essential to prevent and resolve health problems, the quality of crude drug including the active chemical composition is essential for medicinal efficacy. Various types of chemical compounds were found in the aerial parts of L. sibiricus, for example, alkaloid (leonurine),, labdane diterpenoid (sibiricinones, leojaponin, isoleoheterin, leosibiric acids), diterpene lactone (leonotinin, leonotin, dubiin, and nepetaefuran), flavonoid (genkwanin and rutin), polyphenol (chlorogenic acid and caffeic acid), iridoid glycoside (acetylharpagide and ajugoside), phenylpropanoid glycoside (lavandulifolioside and verbascoside), sterol (β-sitosterol and β-sitosterol glucoside), etc., Leonurine [Figure 1] is a highlighted compound in L. sibiricus regarding its pharmacological properties such as neuroprotective effects and cardiovascular protective effects.,, Leonurine or 4-guanidino-n-butyl syringate is a pseudoalkaloid which is acylguanidino derivative of syringic acid and 4-guanidino-n-butanol. To identify the active constituents in medicinal plants, chromatographic techniques are world-widely used in various studies. High performance liquid chromatography (HPLC) has been previously reported for the analysis of leonurine in L. japonicus Houtt.,, In addition, capillary electrophoresis (CE) is accepted as a separation technique for plant secondary metabolite analysis, for example, iridoids, phenylpropanoids, flavonoids, lignans, and alkaloids., CE use high-voltage direct current electric field as driving force for analyte separation performed in capillaries and the analysis time or migration time is usually <30 min, faster than HPLC., At present, leonurine quantification in L. sibiricus in Thailand, particularly with the CE technique, has not been reported. Therefore, this study presented the quality parameters of L. sibiricus aerial part crude drug in Thailand with special reference to its leonurine content analyzed by CE.
| Materials and Methods|| |
L. sibiricus dried aerial parts were provided from 15 traditional drugstores located in four regions of Thailand [Table 1]. All crude drugs were authenticated by the expert taxonomist (Nijsiri Ruangrungsi) as well as comparison with the identified herbarium specimens at the Forest and Plant Conservation Research Office, Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Thailand. Microscopic anatomy of the stem was also compared to previous report. Voucher specimens and numbers were deposited at the research institution.
Instruments and chemicals
CE system (Agilent 7100, USA), muffle furnace (Carbolite Gero, UK), digital microscope (Carl Zeiss, Germany), water purification system (Heal Force, China), TLC silica gel 60 GF254 plate (Merck, Germany), leonurine (SML0670, Sigma-Aldrich, USA), and AR grade solvents (RCI Labscan, Thailand).
Determination of pharmacognostic specification
Plant material identification
L. sibiricus flowering branches were visually inspected. The transverse section of the stem was examined under a digital microscope for the anatomical characteristics. The crude drug was pulverized and microscopically investigated for histological characteristics. Those characters were botanically illustrated by hand drawing.
Determination of loss on drying, total ash, and acid-insoluble ash
Three grams of the powdered crude drug were spread in an even layer in a pre-weighed crucible and heated at 105°C for the loss on drying content. After that, the crucible was placed in a muffle furnace for incineration at 550°C for 6–8 h. The crucible containing the total ash was weighed after cooling in a desiccator. Then, 25 ml of hydrochloric acid (70 g/l) was added and boiled gently for 5 min. The insoluble matters were collected on an ashless filter paper and transferred to the original crucible, dried on a hot plate, and incinerated again. After cooling in a desiccator, the crucible containing the acid-insoluble ash was weighed.
Determination of ethanol- and water-soluble extractive matters
Five grams of the powdered crude drug was macerated in 70 ml of 95% ethanol or water for 6 h under shaking then 18 h under standing at room temperature. After filtration, the marc was washed and the volume was adjusted to 100 ml with the solvent used. Twenty milliliters of the filtrate were aliquoted into a pre-weighed small beaker, heated to dryness at 105°C, and weighed. The extractive matters were calculated.
Determination of moisture content
Fifty grams of the powdered crude drug was transferred to the flask containing 200 ml of water-saturated toluene. The flask was connected to the apparatus for azeotropic distillation of water in crude drug and toluene. The volume of water in the receiving tube was then recorded.
Thin-layer chromatographic fingerprint identification
Another one of 20-ml aliquot of the ethanolic filtrate from the extractive matter determination was evaporated and re-dissolved in 0.5 ml of methanol. Five microliters of the extract were applied to TLC plate, developed with the mobile phase of ethyl acetate: methanol: water: formic acid (8.4: 0.6: 0.5: 0.5) and observed under 254 and 366 nm ultraviolet light as well as by spraying with p-anisaldehyde's reagent.
Quantitative analysis of leonurine by capillary electrophoresis
Five grams of each cleaned, dried, and pulverized sample was exhaustively extracted with 95% ethanol (200 mL) by the Soxhlet apparatus (6 h). After filtration, the filtrate was evaporated to dryness. The extract was weighed to calculate the % yield and then dissolved in methanol to obtain the final concentration (1 mg/1 mL). The extract in methanol was diluted to various concentrations and filtered through a 0.45 μm PTFE membrane syringe filter for further CE analysis.
Standard preparation and calibration curve of leonurine
The stock standard solution of leonurine was prepared by dissolving the leonurine (1 mg) in methanol (1 mL). The solution was filtered through a 0.45 μm PTFE membrane syringe filter. The standard stock solutions of leonurine (20-100 μg/mL) were applied in triplicate on a CE column. The resolved peak area was recorded for each of the standard concentration. The calibration curve of leonurine was plotted by taking peak area versus concentrations of standard.
Capillary electrophoresis conditions
CE system comprised of Agilent 7100 CE equipped with auto-sampler and photodiode array (PDA) detector. The condition was set at 20°C, and applied voltage of 25 kV. The detection was performed at 277 nm. All filtered samples and standard were injected 5 s from each vial. The buffer solution was 20 mM sodium phosphate buffer, pH 7.9. Capillary column was uncoated fused silica capillary 64.5 cm length (56 cm to the detector) × 50 μm i. d. The leonurine content of each sample was determined by comparing the peak area of standard leonurine with a calibration curve. All data acquisition was performed using HP ChemStation software, version B.04.03.
According to ICH guidelines, specificity, linearity, limit of detection and limit of quantification, precision, accuracy, and robustness were checked for confirming method validation. All the parameters were performed in triplicates.
The specificity of the method was determined by analyzing the absorbance spectra of standard leonurine and samples. Peak purity was evaluated by comparing its peak at peak start, peak apex, and peakend position.
The linearity of the method was expressed as a calibration range generated by plotting peak areas versus concentrations of standard leonurine. The coefficient of determination (R2) was calculated using Excel software.
Limit of detection and limit of quantification
LOD, the lowest amount that can be detected, and LOQ, the lowest amount that can be quantitated, were calculated from the calibration curve using the following formula:
Where residual SD = the residual standard deviation of the regression line, S = the slope of the regression line.
The repeatability and intermediate precision were assessed by analyzing the sample solution with three concentrations (low, medium, and high) (each in triplicate) on the same day and three different days, respectively. The precision of leonurine content analysis was determined in terms of percent relative standard deviation (% RSD) by the following equation:
The percent recovery was determined by adding different level concentrations of standard leonurine to a pre-analyzed sample. The analysis was done by the proposed CE method and the analysis was carried out in triplicate.
Where A = the amount of leonurine found in the spiked extracted sample
B = the amount of leonurine found in the un-spiked extracted sample
C = the amount of standard leonurine was added to the extracted sample
A small variation on the pH buffer solution from 7.8 to 8.0 was applied for the robustness. The result was expressed as % RSD.
| Results and Discussion|| |
Pharmacognostic specification of Leonurus sibiricus aerial part
The drawing of a flowering branch and photograph of the dried crude drug (aerial parts) of L. sibiricus are shown in [Figure 2] and [Figure 3]. The transverse section of the stem of L. sibiricus showed anatomical characters of collenchyma, epidermis, glandular trichome, parenchyma, prism crystal, xylem fiber, and xylem vessel [Figure 4]. The quadrangular stem highlighted the ribs with angular collenchymatous cells was illustrated. The epidermis was uniseriate with abundant glandular trichomes distribution. Prismatic calcium oxalate prisms were found in the stem medullary parenchyma. The microscopic anatomy of L. sibiricus stem in Thailand was found to be unique as L. sibiricus stem reported in Brazil. The histological evaluation of powdered crude drug showed a bordered pitted vessel, fragment of fiber, fragment of the lower epidermis, glandular trichome, prism crystal, parenchyma in longitudinal view, parenchyma in transverse view, spiral vessel, starch granule, and stomata [Figure 5]. The physicochemical parameters due to the quality of L. sibiricus are shown in [Table 2]. The loss on drying, total ash, acid-insoluble ash, and moisture contents should be not more than 8.18, 15.28, 4.04, and 8.91% by dry weight, whereas ethanol and water-soluble extractive values should be not <7.67 and 17.21 g/100 g of dry weight, respectively. TLC fingerprint iss shown in [Figure 6]. The total and acid-insoluble ashes were higher than previous study of L. sibiricus crude drugs in Korea in 2001 which reported loss on drying, total ash, and acid-insoluble ash as 7.94% ±0.63%, 8.51% ±2.1%, and 1.24% ±0.77%, respectively.
|Figure 4: Transverse section of the stem of Leonurus sibiricus. (1) Glandular trichome, (2) collenchyma, (3) xylem fiber, (4) epidermis, (5) prism crystal, (6) xylem vessel, (7) parenchyma|
Click here to view
|Figure 5: Histological characters of Leonurus sibiricus aerial part powder. (1) Parenchyma in transverse view, (2) parenchyma in longitudinal view, (3) spiral vessel, (4) bordered pitted vessel, (6) glandular trichome, (7) fragment of lower epidermis, (8) prism crystal, (9) starch granule, and (10) fragment of fiber|
Click here to view
|Table 2: The physicochemical parameter due to quality of Leonurus sibiricus|
Click here to view
|Figure 6: TLC of methanolic extract of Leonurus sibiricus. Solvent systems; ethyl acetate: methanol: water: formic acid 8.4:0.6:0.5:0.5. Detections; I = detection under UV light (254 nm). II = detection with UV light (366 nm). III = detection with p-anisaldehyde's reagent|
Click here to view
Leonurine content in Leonurus sibiricus aerial part
The ethanolic extraction using Soxhlet apparatus of L. sibiricus crude drugs from 15 locations yielded 18.86 ± 4.09% dry weight [Table 1]. CE coupling with PDA detector was used for quantitative analysis of leonurine in the extracts in this study due to several advantages, i.e. high separation efficiency, short analysis times, low waste generation, and a diverse range of applications. The optimized condition was developed and the method was validated in terms of linearity, accuracy, precision, LOD, LOQ, specificity, and robustness [Table 3]. The electropherogram of the ethanolic extract of L. sibiricus is shown in [Figure 7]. Leonurine migration time was 6.2 min. The absorbance spectrum of leonurine scanned from 200 to 700 nm with the maximum absorption at 277 nm is shown in [Figure 8]. The identity of the spectra of standard and sample leonurine [Figure 8] as well as peak purity index of 0.9999 [Figure 9] represented the specificity of the method. The calibration curve showed linearity in the range of 20–100 μg/mL with the regression equation of y = 0.0871x + 0.9537 and the coefficient of determination (R2) was 0.9995 [Figure 10]. The accuracy was confirmed by spiking standard leonurine at three different concentrations (10, 30, and 50 μg/mL) into L. sibiricus extracted sample. The result was found to be 96.39%–101.83% recovery (average 99.54% recovery) [Table 3]. The average values of repeatability and intermediate precisions were 2.97 and 3.16% RSD, respectively. The limit of detection and the limit of quantitation, calculated from the residual standard deviation of the regression line and the slope of the calibration curve, displayed 1.59 and 4.82 μg/mL, respectively. The robustness was performed by varying the pH buffer solution from 7.8–8.0, and the average result was found to be 2.38% RSD [Table 3]. The developed CE was valid for the quantification of leonurine in L. sibiricus ethanolic extract. [Table 1] shows the contents of leonurine in 15 L. sibiricus crude drugs that ranged from 0.79 to 4.23 mg/g with the average of 2.38 ± 1.10 mg/g or 0.24% ±0.11%. The amount of leonurine in L. sibiricus crude drug in this study was higher than the previous report of L. sibiricus crude drug in Korea that demonstrated the leonurine content analyzed by HPLC as 0.124% ±0.066%. In addition, leonurine found in this study seemed to be higher than leonurine in L. japonicus crude drug in China quantitated by HPLC-MS/MS that was found to be 0.73 to 2.33 mg/g. This finding revealed the beneficial application of CE technique for quality evaluation of medicinal plant material with reference to its bioactive phytochemicals.
|Table 3: Method validation parameters of leonurine in Leonurus sibiricus extract|
Click here to view
|Figure 7: Capillary electrophoresis Chromatogram of ethanolic extract of Leonurus sibiricus L. at 277 nm|
Click here to view
|Figure 8: The absorption spectra of standard leonurine (a), and sample (b)|
Click here to view
|Figure 9: Peak purity of leonurine in Leonurus sibiricus extract (peak purity index: 0.9999, peak start at 6.143 min and end at 6.350 min)|
Click here to view
| Conclusion|| |
The pharmacognostic specification of L. sibiricus in Thailand was established. The microscopic characteristics, including the transverse section of the stem and the powder characters of L. sibiricus, were illustrated. The TLC fingerprint and physicochemical parameters were demonstrated by this study and could be useful for the identification of L. sibiricus dried crude drug. The CE equipped with a PDA detector was developed, validated, and performed for quantification of leonurine in L. sibiricus. The leonurine contents in L. sibiricus from various areas in Thailand were revealed which could be used for the specification of this crude drug concerning its chemical marker.
Financial support and sponsorship
College of Public Health Sciences, Chulalongkorn University.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Leonurus sibiricus Linnaeus. Flora of China 1994; 17:162–165. [cited 2021 Mar 26]. Available from: http://efloras.org/
Kanchanapoo J, Kaewamatawong R, Khamdang S, Chinsai K, Ubolwat S, Jandee K. Effects of Thai herbs used in traditional women remedies on the uterine contraction. Thai Pharm Health Sci J 2011;6:202-8.
Jaroenngarmsamer P, Ounprasertsuk J, Krutchangthong P, Dumklieng W. Herbal, postpartum care in Thai traditional medicine. Proceedings of the International Academic Multidisciplinary Research Conference; 2019 Mar 7-8; London, pp 292-7.
Leangpanich S, Itharat A, Chanvimalueng W, Mukkasombat N. A preliminary study on efficacy of Prapchompoothaweep remedy for treatment of allergic rhinitis patients and their quality of life after the treatment. Thammasat Med J. 2019;19:537-46.
Onthong N, Chonpatathip U, Rajanivat Y, Patthananurak K, Sangvichien S, Kamoltham T. A comparative study on the effects of Prabchompoothaweep remedy and loratadine in treatment of patients with allergic rhinitis and upper respiratory tract infections at Pathumtani hospital. TH J of Health Edu. 2019;42:135-45.
Hayashi Y. Studies on the ingredients of Leonurus sibiricus L. II. Structure of leonurine.(2). Yakugaku Zasshi. 1962;82:1025-7.
Zachow LL, Ávila JM, Saldanha GA, Mostardeiro MA, da Silva UF, Morel AF, et al. Chemical composition and evaluation of prolyl oligopeptidase and acetylcholinesterase inhibitory activities of Leonurus sibiricus L. from Brazil. Nat Prod Res. 2017; 31:1459-63.
Boalino DM, McLean S, Reynolds WF, Tinto WF. Labdane diterpenes of Leonurus sibiricus. J Nat Prod. 2004;67:714-7.
Narukawa Y, Niimura A, Noguchi H, Tamura H, Kiuchi F. New diterpenoids with estrogen sulfotransferase inhibitory activity from Leonurus sibiricus L. J Nat Med. 2014; 68:125-31.
Satoh M, Satoh Y, Isobe K, Fujimoto Y. Studies on the constituents of Leonurus sibiricus L. Chem Pharm Bull. 2003;51:341-2.
Pitschmann A, Zehl M, Heiss E, Purevsuren S, Urban E, Dirsch VM, et al. Quantitation of phenylpropanoids and iridoids in insulin-sensitising extracts of Leonurus sibiricus L. (Lamiaceae). Phytochem Anal. 2016;27:23-31.
Yang D, Jia W, Zhu YZ. Leonurine, a potential agent of traditional Chinese medicine: recent updates and future perspectives. Nat Prod Commun. 2016;11:1757-61.
Zhu YZ, Wu W, Zhu Q, Liu X. Discovery of Leonuri and therapeutical applications: from bench to bedside. Pharmacol Ther. 2018;188:26-35.
Huang L, Xu DQ, Chen YY, Yue SJ, Tang YP. Leonurine, a potential drug for the treatment of cardiovascular system and central nervous system diseases. Brain Behav. 2021;11:e01995.
Yeung H, Kong Y, Lay W, Cheng K. The structure and biological effect of leonurine. Planta Med. 1977;31:51-6.
Tan YJ, Xu DQ, Yue SJ, Tang YP, Guo S, Yan H, et al. Comparative analysis of the main active constituents from different parts of Leonurus japonicus Houtt. and from different regions in China by ultra-high performance liquid chromatography with triple quadrupole tandem mass spectrometry. J Pharm Biomed Anal. 2020; 177: 112873.
Chen Z, Wu JB, Liao XJ, Yang W, Song K. Development and validation of an UPLC-DAD-MS method for the determination of leonurine in Chinese motherwort (Leonurus japonicus). J Chromatogr Sci. 2010;48:802-6.
Xie J, Sang L, Zhang Y, Fang L, Li Y. Determination of stachydrine and leonurine in Herba leonuri and its succedaneum-”Herba lagopsis-”with a sensitive HPLC–MS/MS method. J Liq Chromatogr Relat Technol. 2015;38:810-5.
Gomes AF, Ganzera M, Schwaiger S, Stuppner H, Halabalaki M, Almeida MP, et al. Simultaneous determination of iridoids, phenylpropanoids and flavonoids in Lippia alba extracts by micellar electrokinetic capillary chromatography. Microchem J. 2018;138:494-500.
Liang J, Gong FQ, Sun HM. Simultaneous separation of eight lignans in Forsythia suspensa by β-cyclodextrin-modified capillary zone electrophoresis. Molecules. 2018;23:514.
Hou Z, Sun G, Guo Y, Yang F, Gong D. Capillary electrophoresis fingerprints combined with linear quantitative profiling method to monitor the quality consistency and predict the antioxidant activity of alkaloids of Sophora flavescens. J Chromatogr B. 2019; 1133:121827.
Wang J, Jiang Y, Wang B, Zhang N. A review on analytical methods for natural berberine alkaloids. J Sep Sci. 2019; 42(9): 1794-815.
Liu X, Jiang W, Su M, Sun Y, Liu H, Nie L, et al. Quality evaluation of traditional Chinese medicines based on fingerprinting. J Sep Sci. 2020;43:6-17.
Duarte MR, Lopes JF. Morfoanatomia Foliar e Caulinar de Leonurus sibiricus L., Lamiaceae. Acta Farm Bonaer. 2005;24:68-74.
Pitakpawasutthi, Y, Palanuvej C, and Ruangrungsi N, Quality evaluation of Kaempferia parviflora rhizome with reference to 5, 7-dimethoxyflavone. J Adv Pharm Technol Res. 2018;9:26–31.
The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology Q2 (R1). Geneva (switzerland): ICH; 2005.
Hong SS, Hwang JS, Lee SA, Hwang BY, Ha KW, Ze KR, et al. Isolation and quantitative analysis of leonurine from Leonuri herba. Korean J Pharmacogn 2001;32:322-6.
Toraño JS, Ramautar R, de Jong G. Advances in capillary electrophoresis for the life sciences. J Chromatogr B. 2019;1118: 116-36.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
[Table 1], [Table 2], [Table 3]