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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 4  |  Issue : 2  |  Page : 59-66

Standardization of Rasagandhi mezhugu: A traditional siddha higher order herbomineral formulation


1 Central Council for Research in Siddha (CCRS), Chennai, Tamil Nadu, India
2 Siddha Regional Research Institute, Central Council for Research in Siddha (CCRS), Poojapura, Kerala, India
3 Siddha Central Research Institute (SCRI), Central Council for Research in Siddha (CCRS), Chennai, Tamil Nadu, India

Date of Submission07-Apr-2022
Date of Acceptance27-Aug-2022
Date of Web Publication23-Jan-2023

Correspondence Address:
Dr. Viswan Lilly Reena
Siddha Regional Research Institute, Poojapura P.O., Thiruvananthapuram - 695 012, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrsm.jrsm_11_22

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  Abstract 

Background: Rasagandhi mezhugu (RGM) is the higher order Siddha formulation comprising of mercury, arsenical compounds, minerals and herbs and is indicated for chronic diseases like Megarogam, Kiranthi, Puttrunoi, etc.
Objectives: The objective of the present investigation was to standardize the formulation of RGM.
Materials and Methods: The organoleptic characters such as color, appearance, taste, and odor were noted. RGM was screened for moisture content, total ash value, water-soluble ash, acid-insoluble ash, alcohol-soluble extractive value, water-soluble extractive value, microbial load, and specific pathogen to estimate the quality of the study drug.
Results: The outcomes of the physiochemical analysis of RGM were found to be within standard limits. RGM is free from specific pathogens such as Escherichia coli, Salmonella, Pseudomonas, and Staphylococcus aureus. The overall safety regarding heavy-metal content was assured by quantifying the metal content using inductively coupled plasma optical emission spectroscopy. The high-performance thin-layer chromatography fingerprint of RGM was generated.
Conclusion: The results attained from the study could be used as a reference for setting limits for the quality assurance and quality control of Rasagandhi mezhugu.

Keywords: Herbomineral, HPTLC, ICP-OES, Rasagandhi mezhugu, Siddha


How to cite this article:
Shyamala R, Reena VL, Lekha GS, Amsaveni S, Sathiyarajeswaran P. Standardization of Rasagandhi mezhugu: A traditional siddha higher order herbomineral formulation. J Res Siddha Med 2021;4:59-66

How to cite this URL:
Shyamala R, Reena VL, Lekha GS, Amsaveni S, Sathiyarajeswaran P. Standardization of Rasagandhi mezhugu: A traditional siddha higher order herbomineral formulation. J Res Siddha Med [serial online] 2021 [cited 2023 Feb 6];4:59-66. Available from: http://www.jrsm.in/text.asp?2021/4/2/59/368437


  Introduction Top


Rasagandhi mezhugu (RGM) is one of the classical Siddha formulations indicated for various diseases, namely Soolai, Kandamaalai, Kiranthi, Vippuruthi, Kurai noi, Mega rogankkal, Puttru noi, Pilavai, and Pautthiram.[1] It is commonly used for cancer, benign tumors, venereal diseases, skin diseases, and neurological ailments. Siddha practitioners prescribe RGM as a therapy for different types of cancers. Some of the studies proved the anticancerous activity of RGM in an animal model.[2],[3],[4]

RGM is a herbomineral compound formulation with mercurial, arsenical compounds, and metals along with 38 herbs.[1],[5] The therapeutic efficacy of RGM is based on the combined action of a mixture of constituents, which provides treatment opportunities for many diseases. According to the Siddha concept, most of the ingredients of RGM have astringent, bitter, pungent, and sour tastes.[6] The taste plays a vital role in balancing the deranged Iyyam in subjects affected and promotes shrinkage of the size of the fibroid.[7]

The presence of heavy metals in RGM formulation requires a regular purification process in order to comply with the stringent quality control measures. Moreover, the raw drugs used should be authenticated before the drug processing. The heavy metals used in RGM are completely transformed into inert compounds through the various step process during the medicine preparation. Furthermore, the toxicities of metals and minerals were nullified by the active principles of the herbs used in purification and processing.

As certain pharmaceuticals are marketing the drug RGM without following the standard operating procedures (SOPs) and using adulterated or spurious raw drugs, there is a need for standardization of the ingredients and the final product RGM. The processing and standardization is exemplified in this article. The present work deals with the processing of RGM as per the SOPs and standardization of Rasagandhi mezhugu. The work involves physicochemical analysis, chromatographic high-performance thin-layer chromatography (HPTLC), fingerprint analysis, quantification of metal content in RGM using inductively coupled plasma optical emission spectroscopy (ICP-OES), and determination of microbial load.


  Materials and Methods Top


All the raw materials were purchased from IMPCOPS, Pvt. Ltd., Chennai, Tamil Nadu, India and authenticated by the Department of Pharmacognosy, Siddha Central Research Institute (SCRI), Chennai, Tamil Nadu, India. Processing as per SOP was carried out in IMPCOPS, Pvt. Ltd., Chennai, Tamil Nadu, India.

Processing of drug

The processing of the drug RGM is well explained in Siddha Literature Pulippani Vaithiyam––500 and The Siddha Formulary of India – Part I (English), 1992, which is enlisted under Drugs and Cosmetics Act 1940. The list of herbal ingredients used along with their botanical name is tabulated in [Table 1].
Table 1: Herbal ingredients in RGM

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The metal ingredients and their chemical names used for the preparation of RGM are tabulated in [Table 2].
Table 2: Metallic ingredients in RGM

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The metallic ingredients of Rasagandhi mezhugu––item nos. 1 to 8 in [Table 2] are purified before the processing of the drug following the standard procedure.

Standard operating procedure of Rasagandhi mezhugu

Stage 1

  • Taken the item nos.1 to 6 in [Table 2], mixed all, and pound into a coarse powder.


  • Ground in the kalvam, made into a fine powder, and dried.


  • The fine black powder obtained from pounding of item nos.7 and 8 in [Table 2] was mixed with above said powder and made into a fine powder


  • Stage 2

  • The herbal ingredients are purified, ground in the pulveriser, and sieved to get fine powder.


  • Stage 3

  • Mixed all the fine powder got from previous stages and broke the country hen’s egg (Kozhimuttai) into the powder.


  • Mixed the white yolk and yellow yolk with the fine powder for 4 to 5 hours thoroughly and made it into a waxy consistency form.


  • Dried and made into the granular form and then ground to fine powder.


  • Stage 4

  • Panaivellam mixed with water, boiled, filtered, and again boiled


  • Mixed the fine powder got from stage 3 with the above mixture when the product attains pakupatham.


  • Dried and made into the granular form and then ground to fine powder.


  • Stage 5

  • Mixed the fine powder got from stage 4 with honey and mixed thoroughly until it attains waxy consistency.


  • The final wax (Mezhugu) should be preserved in the earthen or ceramic bowl.

    Physicochemical standardization

    Physicochemical studies of Rasagandhi mezhugu––typically loss on drying (LOD), total ash, water-soluble ash, acid-insoluble ash, water-soluble extractive, alcohol-soluble extractive, and pH––were carried out as per the standard protocol.[8]

    Determination of microbial load

    Microbial tests were carried out to estimate the number of viable aerobic microorganisms present in the formulation that includes Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, yeasts, molds, and total microbial plate count. The microbial loads were determined as per Ayurvedic pharmacopeia.[9],[10]

    High-performance thin-layer chromatography analysis

    HPTLC fingerprinting for RGM was done using CAMAG (Switzerland) HPTLC. 4 g RGM in 100mL ethanol was subjected to Soxhlet extraction. Filtered extract was concentrated. 10 µL of each of the extract was applied on silica-coated thin-layer chromatography (TLC) plate (60 F254) using CAMAG ATS4 applicator and developed in a Twin trough chamber (CAMAG) (10 cm × 10 cm), previously saturated with the mobile phase toluene: ethyl acetate: formic acid (8:2:0.1 v/v/v). The plate was developed up to 85 mm from the bottom. It was photo-documented using CAMAG TLC visualizer under short ultraviolet (UV) and long UV. Scanning was performed by TLC Scanner 4 for λ 254 and λ 366 nm. The scanned plates were derivatised with vanillin sulphuric acid and heated at 130ºC for the better visibility of bands. Photographs were taken and the plates were further scanned at λ 540 nm.

    Inductively coupled plasma optical emission spectroscopy analysis

    ICP-OES analysis was done using Agilent 5100 vertical dual view instrument used with the following operation conditions: an radio frequency (Rf) power 1.2 kW, a plasma gas flow rate of 12 L min–1, and a nebulizer gas flow rate of 0.70 L min–1. Sample preparation was carried out by digesting 50 mg of the sample with 1 mL of ultrapure nitric acid using Anton Paar microwave digestion unit and making up to 50 mL. The calibration standard solution is prepared for 0.25 to 10 µg/mL by using ultrapure nitric acid and blank also. The samples are introduced into the plasma using a nebulizer and spray chamber for the analysis of the elements namely Al (396.15 nm), As (188.98 nm), Ba (614.17 nm), Ca (396.85 nm), Cd (214.44 nm), Co (238.89 nm), Cr (266.60 nm), Fe (238.20 nm), K (766.49 nm), Mn (257.61 nm), Mo (379.83 nm), Ni (227.88 nm), Pb (220.35 nm), Se (196.03 nm), Sr (407.77 nm) and Zn (213.86 nm), and Hg (194.164).


      Results and Discussion Top


    Organoleptic properties of Rasagandhi mezhugu justifies the authenticity of the finished formulation with respect to its dark brown color, sweet, and sour taste by its identity as described by Siddha literature. Physicochemical parameters––LOD, total ash, acid-insoluble ash, water-soluble ash, water, and alcohol-soluble extractive, and pH values were further determined to check the purity of the samples. The results of physicochemical parameters are summarized in [Table 3].
    Table 3: Physicochemical parameters of samples

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    Physicochemical parameters

    Physicochemical analysis revealed the identity and purity of RGM. High-moisture content in any drug enhances the microbial growth and hence reduces its shelf life. Recording the moisture content is therefore a crucial step to be followed. The moisture content of RGM was less than 3% pertaining to a little microbial growth as well as sticking trouble in the final processing. Total ash value gives an idea about the percentage of inorganic content in them. It measures the total amount of material remaining after ignition. It includes both physiological ash which is derived from the plant tissue and non-physiological ash which is the extraneous matter such as sand and soil adhering to the plant surface and also from minerals added. The total ash value was calculated to be ~7%. The water-soluble salts in RGM, which was accounted by water-soluble ash value, were calculated to be 2.57%.

    Acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid and igniting the remaining insoluble matter. This measures the amount of silica present, especially in sand and siliceous earth. The acid-insoluble ash was found to be 1.22% constituted by siliceous material. The water and ethanol soluble extractive values were estimated to be 56.77% and 56.33%, respectively, indicating the better dissolution of the drug which can lead to its better adsorption in the body. This will give a better therapeutic effect on low dosage of the medicine. The total sugar in RGM is calculated to be 18.75%.

    Microbial analysis

    Microbial contaminants in the drug pose a potential risk for one's health and attention should be taken to assure safety. Total microbial count and aflotoxin content were analyzed and are tabulated in [Table 4]. RGM is free from specific pathogen such as E. coli, Salmonella, Pseudomonas, and Staphylococcus aureus.
    Table 4: Analysis of total microbial count

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    Inductively coupled plasma optical emission spectroscopy analysis

    Qualitative and quantitative metal analysis was carried out using ICP-OES. [Figure 1] shows the overlayed plot, the linearity curve, and the sample curve of the representative metals such as mercury, arsenic and lead, respectively. The results of other elements are given in supporting information [Figure S1] [Additional file 1]. Analysis of the samples was performed in triplicate and the average content of mercury (Hg), arsenic (As), and lead (Pb) was found to be 1.87, 0.44, and 0.56 (% w/w), respectively. The results obtained are tabulated in [Table 5].
    Figure 1: Inductively coupled plasma optical emission spectrometry analysis of RGM

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    Table 5: Elemental analysis of RGM

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    The presence of metals such as arsenic, mercury, iron, copper, and lead are observed in a slightly higher percentage and was undeniably in coordination with the inorganic ingredients added during the preparation of drug. The other metals such as molybdenum, selenium, cobalt, chromium, and cadmium are present below detection limit which ensures the purity and safety of the drug, RGM. It therefore substantiates the accurate and finest processing of the drug. Oxidation state of the metal is one of the important factors to be considered for safety of the drug. As far as no adverse effects have been reported while prolonged use of the drug clinically, indirectly indicating the safety of metallic preparations.

    High-performance thin-layer chromatography analysis

    HPTLC fingerprint of RGM was recorded using Camag HPTLC instrument. HPTLC is one of the sophisticated instrumental techniques which escalate the quality control of a drug one step ahead.[11] The fingerprint of a drug will be the same under identical conditions which makes chromatographic fingerprinting a perfect method for identification and authentication of herbal drugs.[12],[13],[14] The photo documentation of ethanol extract of the sample under UV chamber at 254 nm, 366 nm, and after derivatization at 575 nm is presented in [Figure 2]. Track 1 and 2 represents 5 µL and 10 µL of sample application. Ethanol extract of RGM showed six bands with Rf 0.15, 0.25, 0.29, 0.38, 0.62, and 0.84 under short UV, nine bands with Rf 0.1, 0.21, 0.26, 0.29, 0.43, 0.50, 0.56, 0.60, and 0.83 under long UV and six bands with Rf 0.41, 0.50, 0.59, 0.79, 0.87, and 0.99 under white light after derivatizing with vanillin sulfuric acid. The chromatograms for a representative track at 254, 366, and 540 nm are displayed in [Figure 3][Figure 4][Figure 5].
    Figure 2: HPTLC profile of RGM

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    Figure 3: HPTLC chromatogram of RGM at 254 nm

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    Figure 4: HPTLC chromatogram of RGM at 366 nm

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    Figure 5: HPTLC chromatogram of RGM at 575 nm

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    The peaks observed can be attributed to the phenols, flavonoids, glycoside, saponins, terpenoids, glycosides, and alkaloids present as major constituents in various herbal ingredients.[13] The HPTLC photographs and chromatogram recorded holds to be the authentic fingerprint for the particular batch of RGM preparation.


      Conclusion Top


    Rasagandhi mezhugu was characterized based on the physicochemical and chromatographic parameters. The outcomes of the physiochemical analysis of RGM were found within standard limits. RGM is free from specific pathogens such as E. coli, Salmonella, Pseudomonas, and Staphylococcus aureus. The overall metal content in the formulation is also standardized. The HPTLC photographs and chromatogram recorded holds to be the authentic fingerprint for the particular batch of RGM preparation. The results attained from the study could be used as a reference for setting limits for the quality assurance and quality control of Rasagandhi mezhugu.

    Acknowledgement

    The authors thank Dr. K. Kanakavalli, Director General, Central Council for Research in Siddha (CCRS) for her support throughout the work. Dr. R. Shakila, Head of the chemistry department, Siddha Central Research Institute (SCRI), CCRS for the facilities provided.

    Financial support and sponsorship

    Not applicable.

    Conflicts of interest

    There are no conflicts of interest.





     
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    Anonymous. Siddha Formulary of India – Part I (English). 1st ed. New Delhi: Department of ISM & H, Govt. of India, Ministry of Health and Family Welfare; 1992. p. 68-9.  Back to cited text no. 1
        
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    Anvarbatcha R, Periasamy VS, Preethy P, Ali AA, Akbarsha MA. Chloroform extract of Rasagenthi Mezhugu, a siddha formulation, as an evidence-based complementary and alternative medicine for HPV-positive cervical cancers: Evidence-based complementary and alternative medicine. Evid Bas Compl Alter Med 2012; 2012:136527.  Back to cited text no. 2
        
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    Baskaran J, Gnanamani A, Stanley VA, Stanley SA. Studies on the anticancer potential (GI50) of the siddha formulation, Rasagenthi Mezhugu on human cell lines. J Chem Pharm Res 2017; 9:1-4.  Back to cited text no. 3
        
    4.
    Baskaran J, Gnanamani A, Stanley VA, Stanley SA. In vivo preclinical evaluation of Rasagenthi Mezhugu, a siddha formulation, on anti-cancer potential in BALB/c mice with CT26 murine colon carcinoma. Indo Am J Pharm 2017;3:266-77.  Back to cited text no. 4
        
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    Sudha V, Ramu L, Niraimathi KL, Savariraj S, Pemaiah B. Chemical standardization studies on Rasaganthi Mezhugu. Int J Pharm Pharmaceut Sci 2012;4:21-6.  Back to cited text no. 5
        
    6.
    Murugesa Mudaliyar CS. Materia Medica (Mooligaivaguppu), Part I. 3rd ed. Chennai: Tamil Nadu Siddha Council; 1988. p. 22, 27, 46, 75, 86, 113, 128, 135, 161, 169, 210, 249, 317, 327, 334, 347, 361, 372, 376, 391, 408, 437, 585, 595, 630.  Back to cited text no. 6
        
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    The Ayurvedic Pharmacopoeia of India. New Delhi: Ministry of Health and Family Welfare, Dept. of ISM & H. Govt. of India, AYUSH; 2016; Part-1; IX. p. 117-30.  Back to cited text no. 9
        
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    Baragi CU, Baragi CP, Vyas KM, Shukla VJ. Standardization and quality control parameters of Dashanga Kwatha Ghana tablet: An Ayurvedic formulation. Int J Ayur Res 2011;2:42-7.  Back to cited text no. 11
        
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    Sharma RK, Arora R. Herbal Drugs: A Twenty First Century Perspective. 1st ed. New Delhi: Jaypee Brothers and Medical Publishers; 2006. p. 40.  Back to cited text no. 12
        
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    Agarwal A, Murali B. Quality assessment of selected medicinal plants. 1st ed.2010;1:193-96.  Back to cited text no. 13
        
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        Figures

      [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
     
     
        Tables

      [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



     

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