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ORIGINAL ARTICLE
Year : 2020  |  Volume : 13  |  Issue : 5  |  Page : 498-511  

Different diagnostic modalities for the study of Kumari (Aloe vera (l.) burm.) Swarasa (Juice) Bhavita Trivanga Bhasma with respect to baseline microbial profile


1 Department of Rasashastra and Bhaishajya Kalpana, Institute for Post Graduate Teaching and Research in Ayurveda, Jamnagar, Gujarat, India
2 Head, Microbiology Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
3 Department of Rasashastra and Bhaishajya Kalpana, IPGT and RA, Gujarat Ayurved University, Jamnagar, Gujarat, India
4 Head Pharmacognosy, IPGT and RA, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India

Date of Submission18-Jul-2019
Date of Decision25-Jun-2020
Date of Acceptance25-Jun-2020
Date of Web Publication7-Sep-2020

Correspondence Address:
Pravin Jawanjal
Department of Rasashastra and Bhaishajya Kalpana, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar - 361 008, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mjdrdypu.mjdrdypu_209_19

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  Abstract 


Background:
In Ayurveda, metal-based preparations, that is Bhasma (incinerated Rasaushadhi), are indicated for the treatment of several diseases. Trivanga Bhasma is a classical Ayurvedic preparation containing Bhasmas of three metals, namely Vanga (Tin), Naga (Lead), and Yashada (zinc), and it is indicated in Madhumeha (diabetes) and MutraRoga (urinary disorders), Napunasakta (impotency), Vandhyatva (Infertility), Swetapradara (Leucorrhea), Vata-Pittadosa, and as Shaktivardhaka(strength). Aims: The study was carried out the stability of Kumari Swarasa Bhavita Trivanga Bhasma concerning its microbial profile. Materials and Methods: A sample of Kumari Swarasa Bhavita Trivanga Bhasma was prepared and studied to check microbial contamination at regular intervals. Results: Every time, the sample was subjected to the microbiological study from the date of the preparation to the date of the last microbiological study. No contamination was found in the microbiological study. Discussion: Hence, the present study was carried out to observe the stability study of Kumari Swarasa Bhavita Trivanga Bhasma concerning microbial contamination of the sample prepared and preserved in different climatic and temperature conditions. Thus, a baseline microbial profile was studied at regular intervals. At the end of the study, it was found that the sample was not shown the presence of any microbes. Conclusion: In the microbiological study of Kumari Swarasa Bhavita Trivanga Bhasma, there was no growth found of microorganisms (bacterial or fungal), from the date of preparation, shown shelf life for 296 days. Hence, in the present study, the stability test of Kumari Swarasa Bhavita Trivanga Bhasma concerning microbiological findings was negative at room temperature, warm and cold, dry, and humid conditions.

Keywords: Bhavita, Kumari, Microbial, Swarasa, Trivanga Bhasma


How to cite this article:
Jawanjal P, Cholera M S, Bedarkar P, Patgiri B J, Harisha C R. Different diagnostic modalities for the study of Kumari (Aloe vera (l.) burm.) Swarasa (Juice) Bhavita Trivanga Bhasma with respect to baseline microbial profile. Med J DY Patil Vidyapeeth 2020;13:498-511

How to cite this URL:
Jawanjal P, Cholera M S, Bedarkar P, Patgiri B J, Harisha C R. Different diagnostic modalities for the study of Kumari (Aloe vera (l.) burm.) Swarasa (Juice) Bhavita Trivanga Bhasma with respect to baseline microbial profile. Med J DY Patil Vidyapeeth [serial online] 2020 [cited 2020 Sep 29];13:498-511. Available from: http://www.mjdrdypv.org/text.asp?2020/13/5/498/294350




  Introduction Top


Bhasma is one of the dosage forms of Ayurvedic drugs prepared using metals and minerals along with herbs. Trivanga Bhasma is a combination of three metals, namely Vanga (tin), Naga (lead), and Yashada (zinc), and it is indicated in Madhumeha (diabetes) and MutraRoga (urinary disorders), Napunasakta (impotency), Vandhyatva (infertility), Swetapradara (leucorrhea), and as Shaktivardhaka (strength).[1]

Kumari (Aloe vera [L.] Burm. fil. [synonym Aloe. barbadensis Miller]) belongs to the Liliaceae family. It is commonly known as Aloe vera, Gwarpatha, and Ghritkumari. It contains saccharides, anthraquinones, vitamins, enzymes, and minerals. Its constituents are aglycone barbaloin, aloe-emodin AIII, and prototinosaponins AIII. Its active constituents are barbaloin, aglycone, aloe-emodin, pseudoprototinosaponin AIII, and prototinosaponins AII. Kumari showed hypoglycemic activity by stimulation of synthesis or release of insulin from beta-cells of the pancreas. Control of carbohydrate metabolizing enzymes maintains glucose homeostasis. pseudoprototinosaponin AIII and prototinosaponins AIII hypoglycemic effect is due to action on hepatic gluconeogenesis or glycogenolysis.[2] The dried sap of Kumari showed significant hypoglycemic effect clinically as well as experimentally.[3]

Bhavana is processed in which trituration of the solid matter with a liquid media for the particular time with sufficient pressure. As mentioned in Rasa tarangini, volumetrically or gravimetrically equal to the amount of solid or loose enough to the consistency of dough. Liquid media used for Bhavana may increase the therapeutic efficacy of the Bhavita substance by adding the trace elements in Bhasma, by giving Bhavana transformation of the coarse powder to finer state. The amendment of Rule No. 161-B of Drugs and Cosmetic Act 1940 specifies the maximum shelf life or date of expiry of the different dosage form of Ayurvedic drugs. The shelf life of Swarasa Bhavita Bhasma is not mentioned in the Gazette of India[4]Trivanga Bhasma was prepared by the second method of “Rasa tantra and Siddha PrayogaSangraha”. Kumari Swarasa was prepared according to “Sharangadhara Samhita.”[5] The final product was prepared by the trituration of Trivanga Bhasma with Kumari Swarasa. The drug was prepared in RS and BK Department, Institute for Post Graduate Teaching and Research in Ayurveda (IPGT and RA), Jamnagar of Gujarat Ayurved University, Jamnagar. No preservative was added to the test drug.

The present study aimed to check microbial contamination in the finished product at different time intervals at different climatic conditions, temperature, and humidity setups.


  Materials and Methods Top


After approval of the institutional ethics committee IEC of IPGT and RA, Jamnagar letter no PGT/7/A/Ethics/2017-18/3042 dated19/02/2018, the study was initiated.

Drug preparation

Trivanga Bhasma was prepared by the second method of “Rasa tantra and Siddha PrayogaSangraha.”

Method of Jarana

The Shodhita Trivanga (Naga, Vanga, and Yashada in equal quantities) is kept in an iron pan and heated. While it is melting, powders of Haridra powder (CURCUMA LONGA LINN.), Vata (FICUS BENGHALENSIS LINN.) Churna (stem bark powder), Ashwattha (FICUS RELIGIOSA LINN) Churna (stem bark powder), and Chincha (TAMARINDUS INDICA LINN) Churna (stem bark powder) are sprinkled in small quantities and stirred with Vata stick. This process is continued until the melted Trivanga is reduced to powder form.

Method of Marana

The Jarita Trivanga Bhasma is ground in Vata Jata (aerial root) Kwatha (decoction) for 3 h. Flat Chakrika is prepared and dried well. These Chakrika (dough) were kept in Sharava (a shallow earthen dish) Samputa (joining) and Sandhi Bandha (joining of earthen dish with cloth) are made and placed in Electric muffle furnace for Puta (calcination). This process is repeated ten times. After, the Trivanga Bhasma is ground in kumari swarasa for 3 h. Flat Chakrika is prepared and dried well. These Chakrika were subjected for Puta in Electric muffle furnace. This process is repeated ten times. The color of Bhasma will be pale yellow.

The final product was prepared by the trituration of Trivanga Bhasma with Kumari Swarasa. Kumari Swarasa was prepared by according to “Sharangadhara Samhita”. In these manners, a total of 5 batches were prepared.

A sample of Kumari Swarasa Bhavita Trivanga Bhasma was prepared (stored at room temperature) and studied to check microbial contamination at regular intervals. The microbiological study has been carried out in Microbiology Laboratory, IPGT and RA, Jamnagar.

Drug material

The drug was prepared in Rasashastra and Bhaishajya Kalpana, (RS and BK). Department, IPGT and RA, Jamnagar of Gujarat Ayurved University, Jamnagar.

Storage

The finished product was stored in air-tight food-grade, plastic containers stored in the open light area in the department at room temperature. A clean and dry stainless steel spoon was used to take medicine.

Microbial profile

Microbial contamination was assessed by two methods to check any mycological findings and bacteriological findings.

  1. Smear examination


    1. Wet mount/10% KOH preparation
    2. Gram's stain.


  2. Culture study.


    1. Fungal culture
    2. Aerobic culture.


The details of the procedures followed are given below.

Smear examination

Wet mount/10% KOH preparation

To rule out any mycological findings, the following procedure was adopted.

Procedure: Potassium hydroxide (KOH) pellets were added in distilled water to prepare a 10% solution in a clean glass tube and it was mixed well. A clean grease-free glass slide was taken. Then, a drop of the sample was put on it, and freshly prepared 10% KOH was added to it, and after that, the sample was covered with a grease-free cover glass. Then, it was allowed to react for 15–20 min to remove extra debris other than fungus. After that, the cover glass was observed under high-power (×40) lens and the findings were noted down properly.

Gram's stain test

Gram staining is a differential staining technique that differentiates bacteria into two groups, Gram-positive and Gram-negative. The procedure is based on the ability of microorganisms to retain the color of the stains used during the Gram stain procedure. Gram-negative bacteria are decolorized by any organic solvent (acetone or Gram's decolorizer), while Gram-positive bacteria are not decolorized as primary dye retained by the cell and bacteria will remain as purple. After the decolorization step, a counterstain effect found on Gram-negative bacteria and bacteria will remain pink. The Gram stain procedure enables bacteria to retain the color of the stains, based on the differences in the chemical and physical properties of the cell wall.[6]

To rule out any bacteriological findings, the following procedure was adopted.

Procedure for Gram's Stain [Figure 1], [Figure 2], [Figure 3]: The clean grease-free glass slide was taken to prepare dry equal thick preparation, i. e., smear. Then, the smear was fixed by passing 3–4 times over the flame of a Bunsen burner. This fixation kills the vegetative form of microbes, renders them permeable to stain, makes the material stick to the surface of the slide, and prevents autolytic changes. Fixed prepared smear was covered with Gram's crystal violet solution and it was allowed to remain for mentioning time as per the kit procedure. Then, the smear was washed off with tap water to remove excessive reagent. Then, the smear was covered with Gram's iodine solution, and it was allowed to remain for some time. Again, the smear was washed off with tap water to remove excessive reagent. The smear was decolorized with Gram's de-colorizer by holding the slide at a slant position and pour Gram's decolorized acetone from its upper end up to the removal of the color of the primary dye, i.e., Gram's Crystal Violet. Then, the smear was washed off with tap water to remove excessive reagent. After that, the smear was covered with Safranin solution and it was allowed to remain for mentioning time as per the kit procedure. Then, the smear was again washed off with tap water to remove excessive reagent. The smear was blotted and it was allowed to dry. Then, the slide was examined under the oil immersion lens, and the findings were properly noted.
Figure 1: Smear staining procedure

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Figure 2: Smear staining procedure

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Figure 3: Sabouraud dextrose agar base bottle

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A. Fungal culture method:

Respected materials were collected with a sterile cotton swab for inoculation purposes on selected fungal culture media (i.e., an artificial preparation).

  • Name of media: Sabouraud dextrose agar base (SDA), Modified (Dextrose Agar Base, Emmons)
  • Company: HIMEDIA Laboratories Pvt. Ltd.
  • Required time duration: 05–07 days
  • Required temperature: 37°C
  • Use of media: For selective cultivation of pathogenic fungi.


Procedure for fungal culture: In the clinical microbiology laboratory, the culture method was employed in the isolation of organisms (the streak culture method was routinely employed). Then, appropriate selective solid media, i. e., SDA Base, Modified (dextrose agar base, emmons) for inoculation purpose was selected. After that, the selective solid media were dried in hot air oven and the dried medium was allowed to cool before specimen inoculation. Selective samples were inoculated by sterile cotton swab to the surface of well-dried culture media. After the streaking process, the inoculated medium was incubated in an inverted position at 37°C for 5–7 days. After the incubation period, the growth was examined by the naked eye in the form of the colony and the growth was confirmed by performing different related biochemical reactions and different staining procedures. After that, the results were noted down properly

Aerobic culture method

Respected materials were collected with a sterile cotton swab for inoculation purposes on selected aerobic culture media (i.e., an artificial preparation).

  • Name of media: MacConkey Agar (MA) and Columbia blood agar (BA)
  • Company: HIMEDIA Laboratories Pvt. Ltd
  • Required time duration: 24–48 h
  • Required temperature: 37°C
  • Use of media: For selective cultivation of pathogenic bacteria.


Procedure for aerobic culture in the clinical microbiology laboratory: the culture method was employed in the isolation of an organism (the streak culture method was routinely employed). Appropriate solid media, i.e., MA and Columbia BA, were selected for inoculation purposes. Then, selective solid media were dried in a hot air oven and the dried medium was allowed to cool before specimen inoculation. Then, the selected sample was inoculated on the surface of the cool dried medium with a sterile cotton swab to the surface of well-dried culture media. After the streaking process, the inoculated medium was incubated in an inverted position at 37°C for 24–48 h in the incubator under the aerobic condition and 10% carbon dioxide atmospheric condition. After selecting the incubation period, the growth was examined by the naked eye in the form of colonization and growth was confirmed by performing different related biochemical reactions and different related staining procedures. After that, the reports were isolated and noted down properly.


  Results Top


Every time, the samples (in which drug preserved) were subjected to the microbiological study from the date of the preparation to the date of the last microbiological study. No contamination was found in the microbiological study. The results are shown in [Table 1], [Table 2] and [Table 3].
Table 1: Total solid content Kumari Swarasa

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Table 2: Observations of Trivanga Bhasma with Kumari Swarasa Bhavana preserved at room temperature

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Table 3: Observations of Trivanga Bhasma with Kumari swarasa Bhavana preserved at room temperature

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The average value of total solid content of Kumari Swarasa was 7.51.


  Discussion Top


Trivanga Bhasma was prepared by the second method of “Rasa tantra and Siddha PrayogaSangraha.”[7]Kumari swarasa was prepared according to Sharangadhara Samhita.

Bhaavana[8] (wet grinding) grinding of the material completely soaked in prescribed liquid media till the liquid is completely evaporated and the material is dried, which is termed as “Bhaavana.” Sometimes, the material may be soaked and left for drying on its own without grinding. “Shuddha dravya,” the end product of 'Shodhana process, is a basic raw material in the process of “Maarana.” This basic raw material is subjected to wet grinding in the juices or decoctions of prescribed plant material. The wet grinding is stopped when the contents in the grinder are converted into the dough.[9]Bhavana brings the minute particle of material in contact with liquid media and facilitates the impregnation of organic/inorganic contents and inherent specific properties of media to the material. Due to heat produced during grinding, there may be the possibility of the chemical reaction between a material and media and thus desired chemical change in the final product and desired compound can be obtained. During Bhavana of Rasaushadhi (metal/mineral and herb mineral formulations), organic components of the liquid media are transferred to the material. This facilitates conversion of Nirendriya dravya (inorganic material form) to Sendriya dravya (organometallic/organomineral compound form), which is easily assimilable and biologically favorable to the body.[10] After Bhavana with Kumari Swarasa cellular constituent, i.e., parenchyma cell, starch grain, and acular crystal cell are found in the sample, whereas no cellular constituent absorbed in Trivanga Bhasma [Figure 4], [Figure 5], [Figure 6], [Figure 7]. This shows that Bhasma enriches Kumari Swarasa. After Bhavana with Kumari Swarasa, it may be increased potency of the finished product. The microbiological study of Kumari Swarasa shows its stability in the range of a minimum of 1 day and a maximum of 3 days after the preparation of the sample.[11]Kumari Swarasa is used in trituration for Trivanga Bhasma; the present study was carried out to observe the stability study of Kumari Swarasa Bhavita Trivanga Bhasma Trivanga Bhasma to rule out microbial contamination of the sample prepared and preserved in different climatic and temperature conditions. Thus, a baseline microbial profile was studied at a regular interval. At the end of the study, it was found that the sample was not shown the presence of any microbes from day 1 to 296 for Batch A, from day 1 to day 291 for Batch B, day 1 to day 271 for Batch C, day 1 to day 145 for Batch D, and day 1 to day 9 for Batch E. Stability is usually expressed in terms of shelf life, which is the period from when the product is produced until the time it is intended to be consumed or used. Microorganism needs water, humidity, and temperature at suitable environmental conditions to develop in any media, surface, or article.
Figure 4: Before Bhavana Trivanga Bhasma

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Figure 5: After of Bhavana Trivanga Bhasma

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Figure 6: After Bhavana Trivanga Bhasma

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Figure 7: After Bhavana Trivanga Bhasma

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  Conclusion Top


Th data analysis concluded, the microbiological study of Kumari Swarasa Bhavita Trivanga Bhasma showed that the quality of the powder is in a standard condition after completion of the study.

Financial support and sponsorship

The study was supported by the Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Anonymous.. “Rasa tantra and Siddha PrayogaSangraha” part 1, 12th ed. Ajmer. Krishna Gopal Ayurveda Bhawan College. 2012. p. 63.  Back to cited text no. 1
    
2.
Priya P, Nisha T, Sumit S. Antidiabetic herbs and their marketed formulations. Int J Green Herbal Chem 2016;5:215-29.  Back to cited text no. 2
    
3.
Noor A, Gunasekaran S, Manickam AS, Vijayalakshmi MA. Antidiabetic activity of aloe vera and histology of organs in streptozotocin-induced diabetic rats. Curr Sci 2008;94:1070-6.  Back to cited text no. 3
    
4.
The Gazette of India. Extraordinary. Part-II., Sec. 3. New Delhi: The Gazette of India; 2016. Available from: http://egazette.nic.in/WriteReadData/2016/168375.pdf. [Last accessed on 2018 Mar 16].  Back to cited text no. 4
    
5.
Sharangadhara Acharya. Translater Murthy Srikanta K.R. Sharangadhara Samhita. Sec. 1. Ch. 1, Ver 2. Varanasi: Chaukhamba Orientalia. Reprint edition; 2016. p. 51.  Back to cited text no. 5
    
6.
Brown AE. Benson: Microbiological Applications. 8th ed.. USA. The McGraw-Hill Companies; 2001. p. 64.  Back to cited text no. 6
    
7.
Anonymous.. “Rasa tantra and Siddha PrayogaSangraha” part 1, 12th ed. Ajmer. Krishna Gopal Ayurveda Bhawan College. 2012. p. 64.  Back to cited text no. 7
    
8.
Sharangadhara Acharya. Translater Murthy Srikanta K.R. Sharangadhara Samhita. Sec. 1. Ch. 1, Ver 2. Varanasi: Chaukhamba Orientalia. Reprint edition; 2016. p. 63.  Back to cited text no. 8
    
9.
Savrikar SS, Ravishankar B. Introduction to 'Rasashaastra' the Iatrochemistry of Ayurveda. Afr J Tradit Complement Altern Med 2011;8:66-82.  Back to cited text no. 9
    
10.
Shinghadiya RK, Patgiri BJ, Prajapati PK, Nariya M. Impact of Bhavana on drugs formulation its effect on Ekakushtha (psoriasis) in Comparison to Virechana Thesis IPGT and RA, GAU, Jamnagar; 2019. p. 156.  Back to cited text no. 10
    
11.
Jawanjal P, Cholera MS, Bedarkar P, Patgiri BJ. Stability study of Kumari (Aloe vera [L.] burm.) Swarasa (juice) with respect to baseline microbial diagnostic modalities. BLDE Univ J Health Sci 2019;4:60-5.  Back to cited text no. 11
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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