|Year : 2021 | Volume
| Issue : 2 | Page : 113-122
Physicochemical standardisation of the homoeopathic drug Rumex acetosella and its comparison with another homoeopathic drug, Rumex crispus
Bibaswan Biswas1, Nilakshi Dey1, G. V. Narasimha Kumar1, Renu Arya2, Anil Khurana2
1 Drug Standardization Division, Dr. Anjali Chatterjee Regional Research Institute for Homoeopathy, Central Council for Research in Homoeopathy, Kolkata, West Bengal, India
2 Central Council for Research in Homoeopathy, New Delhi, India
|Date of Submission||21-Dec-2019|
|Date of Acceptance||28-May-2021|
|Date of Web Publication||29-Jun-2021|
Dr. Bibaswan Biswas
Dr. Anjali Chatterjee Regional Research Institute for Homoeopathy, Central Council for Research in Homoeopathy, Kolkata - 700 035, West Bengal
Source of Support: None, Conflict of Interest: None
Background: Rumex acetosella, a relatively new drug in the homoeopathic system, is traditionally used for treating inflammation, diabetes and gastrointestinal problems, especially diarrhoea. Objective: The aim of this work is to report physicochemical standardisation of the homoeopathic drug, Rumex acetosella. Further, we propose our simple and economical method to differentiate it with a closely related species, Rumex crispus. Materials and Methods: The physicochemical study measuring several parameters was done. The pH of the mother tincture (MT) and the water extract was measured and compared. Furthermore, the chemical composition of the Rumex acetosella MT were compared with taxonomically closely related Rumex crispus MT by thin layer chromatography (TLC). Results: The results show a strong relationship between the extracting solvent's polarity and its extracting power. A simple TLC study shows a strong correlation between two drugs of the same genus, Rumex, but they can be differentiated by their unique spots. Conclusion: Our study not only provides the physicochemical standards for the drug Rumex acetosella but also shows that a simple analytical technique, a manual TLC can easily be used to distinguish two taxonomically close homoeopathic drugs.
Keywords: Drug standardisation, Homoeopathy, Physicochemical, Rumex acetosella
|How to cite this article:|
Biswas B, Dey N, Kumar GV, Arya R, Khurana A. Physicochemical standardisation of the homoeopathic drug Rumex acetosella and its comparison with another homoeopathic drug, Rumex crispus. Indian J Res Homoeopathy 2021;15:113-22
|How to cite this URL:|
Biswas B, Dey N, Kumar GV, Arya R, Khurana A. Physicochemical standardisation of the homoeopathic drug Rumex acetosella and its comparison with another homoeopathic drug, Rumex crispus. Indian J Res Homoeopathy [serial online] 2021 [cited 2022 May 29];15:113-22. Available from: https://www.ijrh.org/text.asp?2021/15/2/113/319605
| Introduction|| |
The homoeopathic system of medicine is one of the most widely used medical systems. Most of the homoeopathic drugs are of natural origin.,, This makes quality assurance (QA) and quality control (QC) and standardisation of homoeopathic medicines, extremely challenging., However, it is mandatory to maintain the high quality and authenticity of the administered homoeopathic drugs because of its safety and efficacy.,,, Considering the present situation and challenges, the World Health Organization and other premier authorities laid down the guidelines for herbal medicines' safety and effectiveness.,,, The advent of new physicochemical–analytical techniques, e.g. high-performance thin layer chromatography (TLC), high-performance liquid chromatography, nuclear magnetic resonance spectroscopy, liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, ultraviolet–visible (UV-Vis) spectroscopy and Fourier-transform infrared spectroscopy, can ensure in-depth chemical profiling of herbal, including homoeopathic medicines.,,,, However, the employment of these analytical techniques requires a substantial financial budget. Hence, we report a simple, highly specific, fast, yet economical physicochemical standardisation of Rumex acetosella. We hope our work may be utilised for pharmacopoeial standards in the future.
The genus Rumex is known to have numerous medicinal uses.,,, Homoeopathic system rightly recognises the therapeutic value of this genus. At least three species of this genus are used as homoeopathic medicines, e.g. Rumex acetosa, Rumex crispus and Rumex acetosella.,,, The first two have been widely studied by the homoeopathic community. The drug under study is a relatively less studied one in this medical system.,,,,, To the best of our knowledge, there is no earlier report of physicochemical standardisation for the homoeopathic formulation of the drug Rumex acetosella. There is no previous report on the preparation of the drug's formulation as per Homoeopathic Pharmacopoeia of India. Thus, as expected, there is no commercially available mother tincture (MT). However, there are non-homoeopathic extracts available.,, Rumex acetosella is traditionally used for treating inflammation, diabetes and gastrointestinal problems, especially diarrhoea.,, Further studies show its anticancer and antioxidant properties.,, To verify the high specificity of this simple and economical study, we compare the drug, Rumex acetosella, with another homoeopathic drug, Rumex crispus, having the same genus (taxonomically, two species with the same genus are closest).
| Materials and Methods|| |
Collection of the raw drug
The botanical names of the drugs Rumex crispus and Rumex acetosella are same as that of their respective homoeopathic names. The raw drug Rumex acetosella was collected from wild in the flowering stage from Thummanatty Junction, Udhagamandalam, on 15 September 2017 (Voucher number: 9107). The other raw drug Rumex crispus was collected from Thettukal, Udhagamandalam, on 16 December 2014 (Voucher number: 8886). The plants were taxonomically authenticated by the Field Botanist at the Centre of Medicinal Plants Research in Homoeopathy, Emerald, Udhagamandalam. The sun-dried plants were collected for the drug preparation. Then, they were shade-dried for 24 h. This sun- and the shade-dried plants have been used as the raw drug for our study.
Whole plant, except the root of Rumex acetosella, was used for physicochemical study of the raw drug, preparation and standardisation of the homoeopathic drug of the same name. The rhizomes of the plant Rumex crispus were used for the preparation and physicochemical study.
There is no known literature available for the preparation of the homoeopathic MT of Rumex acetosella. Hence, we adopted maximum extractive value (MEV) method to find out the alcohol percentage for the preparation of the MT. The water was distilled twice before using for MT preparation and other studies. For TLC, aluminium plates pre-coated with silica gel and fluorescent indicator 60F-254™ of 0.25 mm thickness manufactured by Merck Chemicals were used. For the detection of spots, UV lamps with 254 nm, 365 nm and visible light were used. In addition, vanillin-sulphuric acid stain was used and observed under visible light.
Procedure for physicochemical study of the raw drug
- Loss on drying (LOD): Around 2 g of accurately weighed air-dried raw drug was kept in a well-ventilated oven for 3 h at 105°C. After removing the drug from the oven, it was allowed to cool to room temperature by keeping the dried raw drug in a desiccator for 20 min. Then, it was again weighed. The LOD (moisture content) in percentage was calculated as follows:
Weight of empty Petri dish More Details = Wempty
Weight of the Petri dish with air-dried raw drug = W+wet
Weight of the air-dried raw drug = W+wet − Wempty = wwet
Weight of the oven-dried (105°C) raw drug = W+dry − Wempty = wdry
- Preparation of MT
As there is no known literature regarding the preparation, we adopted the MEV method to ascertain the alcohol percentage for the MT preparation.
- Extractive values
- Around 2.0 g of the accurately weighed raw drug was taken (moisture content was taken into account). To it, 50 mL of EtOH was added and kept for 24 h at room temperature. After filtering it, 10 mL was taken and evaporated on a water bath to remove most alcohol. It was then heated at 105°C in a well-ventilated oven until a constant weight was achieved by removing the rest of the solvent. The above experiment was performed twice, and the average value was reported. The ethanol extract value was calculated as follows.
Drug weight = wdrug (moisture content was subtracted to calculate the drug weight)
Empty Beaker weight = wempty
Beaker +dried Extract weight = w+drug
- In a closely related experiments, extract values in petroleum ether, chloroform, ethyl acetate and methanol were determined using 50 mL of the chosen solvent instead of ethanol.
- Ash values:
- Determination of total ash value
Around 2.0 g of the accurately weighed raw drug was taken (moisture content was taken into account) in a thermally resistant previously weighed crucible. The crucible, along with its content, was heated to 450°C for 30 min under aerial conditions. The crucible was cooled in a desiccator for 15 min and weighed. This procedure was repeated until a constant weight was obtained.
Then, the percentage of total ash was calculated as follows.
Empty crucible weight = wempty
Drug weight== wdury
Crucible + ash weight = w+ash
Total ash = w+ash − Wempty = wash
The above experiment was performed twice, and the average value was reported.
- Determination of acid-insoluble ash value
The ash remained from the previous experiment (4 a) was boiled with 25 mL of 10% HCl for 5 min. The insoluble matter was collected over an ash-less filter paper, washed with hot water and ignited for 15 min at a temperature of about 450°C under aerial condition. The crucible was cooled in a desiccator for 15 min and weighed. The heating was repeated until a constant weight is reached. The above experiment was performed twice, and the average value was reported.
Empty Crucible weight = wempty
Drug weight== wdrug
Crucible + acid-insoluble ash weight = w + acid (i) ash
Acid-insoluble ash = w+acid (i) ash − wempty
- Determination of water-soluble ash value
The ash remained from the previous experiment (4 a) was boiled with 25 mL of water for 5 min. The insoluble matter was collected on an ash-less filter paper and washed with hot water. The ash-less filter paper and the residue were ignited for 15 min at a temperature of about 450°C under aerial conditions. The crucible was cooled in a desiccator for 15 min and weighed, repeated for a constant value. The weight of insoluble matter was subtracted from the weight of total ash. The difference in weight represents the water-soluble ash. The percentage of water-soluble ash was calculated as follows. The above experiment was performed twice, and an average value was reported.
Empty crucible weight = wempty
Drug weight== wdrug
Crucible + water-insoluble ash weight = w+water (i) ash
Water insoluble ash = w+water (i) ash − wempty
Total ash= w+ash
Water-soluble ash = Total ash − water-insoluble ash = wash – wwater(i) ash = wwater (s)
Procedure for physicochemical study of the homoeopathic formulation (i.e. mother tincture)
- Determination of total solids
A 10 mL of MT of the drug was heated on a water bath to remove the alcohol. After that, the water content was removed by heating it inside a well-ventilated oven. The sample was cooled in a desiccator for 15 min and weighed. The heating process was repeated until a constant value is reached. The above experiment was performed twice, and the average value was reported.
Empty beaker weight = wempty
Beaker + dried extract weight = w+dried drug
Total solids = w+dried drug – wempty
Specifically, in this case:
- Weight per mL = 10 mL of the MT was weighed, and the weight was divided by 10 to get the data
- The pH was determined by a digital pH-meter. The sample's pH was recorded only after calibration using buffer solutions (pH = 4 and pH = 9.2) each time
- λmax: The measurement was made by diluting the MT by ~ 100 times. The diluting solvent is the same as the MT solvent system
- LC: Around 20 mL of the MT was heated on a water bath to remove the alcohol. The leftover, thus obtained, was extracted by three 20 mL portions of chloroform. The chloroform extract was then concentrated to ~ 2 mL by heating on a water bath. This concentrated extract was used to carry out TLC on pre-coated silica gel aluminium plate 60F-254 of 0.25 mm thickness manufactured by Merck, using a 9:1 CHCl3:CH3OH solvent system
For other solvents, i.e. petroleum ether, ethyl acetate, chloroform, ethanol and methanol, 1.00 g of the plant material was extracted with the chosen solvent. Then, the extract obtained after filtration was concentrated to 2 mL. That concentrated extract was used directly for TLC study.
| Results and Discussion|| |
Rumex acetosella is a perennial herb with a lean and reddish erect stem. The branching happens at the top. The plant is around 50 cm in height. The leaves are arrow-shaped. The taxonomic classification of Rumex acetosella is as shown in [Figure 1].
The raw drug's experimental physicochemical parameters are tabulated in [Table 1].
The total ash was around 12%–13%. This value indicates the metal, silica and silicates content of the sample. As the root has also been included in the standardisation study, soil particles' contribution to the total ash value cannot be ruled out. This assumption may further be justified by the fact that the sample has considerable acid-insoluble ash value. The extractive values in different solvents show a strong correlation between the solvent's polarity and the extractive values. With the increase in the solvent's polarity, there is an increase in extractive values [Figure 2]. The overall result has been summarised in the following graph. The moisture content was found to be around 8%–9%. This moderately low value is expected as the plant sample used has already been sun- and shade-dried.
The values of the physicochemical parameters for the finished product/MT are summarised in [Table 2].
The specific gravity of the prepared MT was 0.94. Considering the solvent's density (~0.90), it is quite effective to extract chemicals from the plant [Table 2]. This observation was re-verified from high total solid, 3.54% w/v of the MT [Table 2]. These values suggest that the solvent system used is quite effective in extracting the plant's chemical components. The pH of the MT was found to be 5.75 [Table 2]. Hence, H+ concentration in the MT is ~ 10 − 5.75. The pH value indicates that the MT is substantially acidic (pH <7 are considered acidic). The MT's acidic nature is expected as Vitamin C is one of the plant's key components. We further investigated the pH issue by measuring the value of the aqueous extract [Table 2]. The pH of the aqueous extract is much lower than that of MT. Our experimental values show that H+ concentration in the aqueous extract is almost eight times that of the MT. We think that the origin of this observation is the less polarity of the solvent system used for the MT compared to water.
From the UV-VIS spectra, the absorbance was found to be in the region of near UV range [Figure 3]. We have also carried out the UV-VIS absorbance of Rumex acetosella in different extractive liquids [Table 3]. As expected, with a change in the extractive liquid's polarity, the pattern of UV spectra (λmax values) change.
|Table 3: Ultraviolet-visible absorbance of Rumex acetosella in different extractive solvents|
Click here to view
|Figure 3: Ultraviolet-visible spectra of Rumex acetosella mother tincture|
Click here to view
To carry out the initial chemical profiling of the drug, Rumex acetosella, we have performed TLC study on the chloroform extract of the MT as well as raw drug extracts in different solvents, e.g. petroleum ether, ethyl acetate, chloroform and methanol using silica gel as the mobile phase and 9:1 CHCl3:CH3OH as the eluent. The TLC plates of the drug under different lights and stain are shown in [Figure 4], [Figure 5], [Figure 6], [Figure 7], and the Rf values are given in [Table 4].
|Table 4: Rf values of the spots of mother tinctures and extracts in different solvents of Rumex acetosella|
Click here to view
|Figure 4: Thin layer chromatography of Pet-ether extract, ethyl acetate extract, chloroform extract of mother tincture, chloroform extract and methanol extract of the drug Rumex acetosella under 254 nm ultraviolet light. Pet-ether: Petroleum|
Click here to view
|Figure 5: Thin layer chromatography of Pet-ether extract, ethyl acetate extract, chloroform extract of mother tincture, chloroform extract and methanol extract of the drug Rumex acetosella under 365 nm ultraviolet light. Pet-ether: Petroleum|
Click here to view
|Figure 6: Thin layer chromatography of Pet-ether extract, ethyl acetate extract, chloroform extract of mother tincture, chloroform extract and methanol extract of the drug Rumex acetosella under white light. Pet-ether: Petroleum|
Click here to view
|Figure 7: Thin layer chromatography of Pet-ether extract, ethyl acetate extract, chloroform extract of mother tincture, chloroform extract and methanol extract of the drug Rumex acetosella under white light with vanillin-sulphuric acid stain. Pet-ether: Petroleum|
Click here to view
[Figure 4], [Figure 5], [Figure 6], [Figure 7] and [Table 4] show that the number of spots for chloroform extract of the MT is the highest. The number of TLC spots indicates that the solvent system used for the MT preparation is exceptionally effective in extracting the chemicals from the plant materials. This TLC study provides preliminary chemical profiling of the plant. The TLCs with other extractive liquids provide the data that can be used in Homoeopathy and other systems of medicines.
Comparative study of plants of different species of the same genus
We compared the homoeopathic formulations (MT) of the drugs Rumex acetosella and Rumex crispus. We sought to compare the homoeopathic formulations instead of raw drugs as QA and QC of the formulations are more important compared to that of raw drugs for safe and effective administration of the medicines. For a comparative study, we focused chiefly on the TLC study. This is because the TLC study would separate different chemicals in the extract based on their on the chemical nature. These different chemicals can be seen as different spots having different colours under UV-VIS light and with TLC stains. As expected, our TLC study shows [Figure 8], [Figure 9], [Figure 10] and [Table 5] that these two taxonomically closely related plants are also chemically similar (they have many spots of the same Rf values when simultaneously eluted from different spots on the same TLC plate). However, there are unique spots for each of the drugs. These unique spots may be utilised to differentiate the respective mother tinctures.
|Table 5: Comparative thin layer chromatography of chloroform extracts of mother tinctures of Rumex acetosella (left) and Rumex crispus (right)|
Click here to view
|Figure 8: Comparative thin layer chromatography of chloroform extracts of mother tinctures of Rumex acetosella (left) and Rumex crispus (right) under 254 nm ultraviolet light|
Click here to view
|Figure 9: Comparative thin layer chromatography of chloroform extracts of mother tinctures of Rumex acetosella (left) and Rumex crispus (right) under 365 nm ultraviolet light|
Click here to view
|Figure 10: Comparative thin layer chromatography of chloroform extracts of mother tinctures of Rumex acetosella (left) and Rumex crispus (right) under white light with vanillin-sulphuric acid stain|
Click here to view
Remarkably, subtle chemical differences between two homoeopathic formulations prepared from herbs of the same genus could be differentiated by simple, cost-effective TLC analysis.
| Conclusion|| |
This work provides the physicochemical parameters of the homoeopathic formulation of the drug Rumex acetosella. Our study proposes physicochemical standards for this drug. Besides, we demonstrated that TLC could be employed as a cheap and straightforward analytical technique to differentiate between two closely related dugs. We believe our study provides a simple, economical, yet significantly sensitive physicochemical standardisation procedure for the drug Rumex acetosella.
The author would like to thank Dr Suraia Parveen, Officer in Charge, Dr. Anjali Chatterjee Regional Research Institute For Homoeopathy (H), Kolkata, West Bengal.
Financial support and sponsorship
Central Council for Research in Homoeopathy, New Delhi, under Ministry of AYUSH Government of India, supported the study.
Conflicts of interest
| References|| |
Rodrigues-Neto JF, Figueiredo MF, Faria AA. Prevalence of the use of homeopathy by the population of Montes Claros, Minas Gerais, Brazil. Sao Paulo Med J 2009;127:329-34.
Nekratova AN, Kosmodemyanskiy LV. A study of Siberia's medicinal plants used in Homeopathy. Homeopathy 2019;108:54-65.
Sultana A, Mukherjee SK. Usefulness of angiospermic plants in homeopathy system of medicine. Int J Pharm Res Bio Sci 2015;4:291-309.
Zhang J, Wider B, Shang H, Li X, Ernst E. Quality of herbal medicines: Challenges and solutions. Complement Ther Med 2012;20:100-6.
Wai-Ping Y, Hian GC, Hwee-Ling K. Quality control and quality assurance of phytomedicines: Key considerations, methods, and analytical challenges. In: Iqbal R, editor. Phytotherapies: Efficacy, Safety, and Regulation. Shangai: Wiley & Sons; 2015. p. 29.
Da-Costa-Rocha I, Edwards S, Lawrence MJ, Cable C, Heinrich M. Quality and safety of herbal products: Part 1 – New legislation and production. Pharm J 2012;288:685-7.
Fong HH, Pauli GF, Bolton JL, van Breemen RB, Banuvar S, Shulman L, et al
. Evidence-based herbal medicine: Challenges in efficacy and safety assessments. In: Current Review of Chinese Medicine: Quality Control of Herbs and Herbal Material.New Jersey: World Scientific; 2006. p. 11-26.
Mills SY. Safety awareness in complementary medicine. Complement Ther Med 1996;4:48-51.
Barnes J. Quality, efficacy and safety of complementary medicines: Fashions, facts and the future. Part II: Efficacy and safety. Br J Clin Pharmacol 2003;55:331-40.
Organization WHO. Research Guidelines for Evaluating the Safety and Efficacy of Herbal Medicines. Manila: WHO Regional Office for the Western Pacific; 1993. p. 1-86.
Bent S. Herbal medicine in the United States: Review of efficacy, safety, and regulation: grand rounds at University of California, San Francisco Medical Center. J Gen Intern Med 2008;23:854-9.
Calixto JB. Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz J Med Biol Res 2000;33:179-89.
Podolsky SH, Kesselheim AS. Regulating homeopathic products – A century of dilute interest. N Engl J Med 2016;374:201-3.
Rawal RS, Bhatt ID, Garhwal S. Chromatographic and spectral fingerprinting standardization of traditional medicines: An overview as modern tools. Res J Phytochem 2010;4:234-41.
Lazarowych NJ, Pekos P. Use of fingerprinting and marker compounds for identification and standardization of botanical drugs: Strategies for applying pharmaceutical HPLC analysis to herbal products. Drug Inf J 1998;32:497-512.
Patel DK, Patel K, Dhanabal SP. Standardization of Berberis aristata extract through conventional and modern HPTLC techniques. Asian Pacific J Trop Dis 2012;2:S136-40.
Naikodi MA, Waheed MA, Shareef MA, Ahmad M, Nagaiah K. Standardization of the Unani drug – Myristica fragrans
Houtt. (Javetri) – With modern analytical techniques. Pharm Methods 2011;2:76-82.
] [Full text]
Yadav NP, Dixit VK. Recent approaches in herbal drug standardization. Int J Integr Biol 2008;2:195-203.
Liang HX, Dai HQ, Fu HA, Dong XP, Adebayo AH, Zhang LX, et al
. Bioactive compounds from Rumex
plants. Phytochem Lett 2010;3:181-4.
Prakash Mishra A, Sharifi-Rad M, Shariati MA, Mabkhot YN, Al-Showiman SS, Rauf A, et al
. Bioactive compounds and health benefits of edible Rumex
species – A review. Cell Mol Biol (Noisy-le-grand) 2018;64:27-34.
Orbán-Gyapai O, Liktor-Busa E, Kúsz N, Stefkó D, Urbán E, Hohmann J, et al
. Antibacterial screening of Rumex
species native to the Carpathian Basin and bioactivity-guided isolation of compounds from Rumex
aquaticus. Fitoterapia 2017;118:101-6.
Pareek A, Kumar A. Rumex crispus
L. – A plant of traditional value. Drug Discov 2014;9:20-3.
Kumar A. Efficacy of lesser known homeopathic medicines in the treatment of bronchial asthma. Int J Adv Ayurveda Yoga Unani Siddha Homeopath 2020;9:558-62.
Gopi KS. Encyclopedia of Medicinal Plants used in Homoeopathy. Vol. I., Ch. 26. Kandern, AIY publications, 2000. p. 603-95.
Boericke W. Pocket Manual of Homoeopathic Materia Medica and Repertory. India: B. Jain Publishers; 2002. p. 558-9.
Reddy E, Sharma P, Singh C. Bronchial asthma and aspidosperma – A study in 50 patients. Tantia Univ J Homoeopath Med Sci 2020;2:24-34.
Clarke JH. A Dictionary of Practical Materia Medica. Vol. III. B. India: Jain Publishers; 1996. p. 1022-7.
Shale TL, Stirk WA, van Staden J. Screening of medicinal plants used in Lesotho for anti-bacterial and anti-inflammatory activity. J Ethnopharmacol 1999;67:347-54.
Orbán-Gyapai O, Lajter I, Hohmann J, Jakab G, Vasas A. Xanthine oxidase inhibitory activity of extracts prepared from Polygonaceae
species. Phytother Res 2015;29:459-65.
Kilic O, Bagci E. An ethnobotanical survey of some medicinal plants in Keban (Elazığ-Turkey). J Med Plants Res 2013;7:1675-84.
Ozenver N, Saeed M, Guvenalp Z, Demirezer LO, Efferth T. Chrysophanol-and nepodin-8-O-β-D-glucopyranoside from Rumex acetosella
, the cytotoxicity towards drug sensitive and multi-drug resistant T leukaemia cancer cells. Planta Med 2016;82:P388.
Baig H, Ahmed D, Zara S, Aujla MI, Asghar MN. In vitro
evaluation of antioxidant properties of different solvent extracts of Rumex acetosella
leaves. Orient J Chem 2011;27:1509-16.
Isbilir SS, Sagiroglu A. Total phenolic content, antiradical and antioxidant activities of wild and cultivated Rumex acetosella
L. extracts. Biol Agric Hortic 2013;29:219-26.
[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], [Table 4], [Table 5]