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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 16  |  Issue : 4  |  Page : 306-311

Microbial study of Bhallatakarishta formulate by two different liquid media


1 Department of Rasa Shastra and Bhaishajya Kalpana, Indian Institute of Ayurved Research and Hospital, Rajkot, Gujarat, India
2 Department of Rasa Shastra and Bhaishajya Kalpana, ITRA, Jamnagar, Gujarat, India
3 Department of Microbiology, ITRA, Jamnagar, Gujarat, India

Date of Submission06-Aug-2021
Date of Decision18-Dec-2021
Date of Acceptance09-Jan-2022
Date of Web Publication17-Dec-2022

Correspondence Address:
Dharmishtha Bopaliya
Department of Rasa Shastra and Bhaishajya Kalpana, Indian Institute of Ayurved Research and Hospital, Rajkot, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/joa.joa_255_21

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  Abstract 


Introduction: Asava and Arishta (fermented formulations) are unique dosage forms of Ayurveda due to their indefinite shelf-life. The self-generated alcohol of these preparations is potentiating both the level pharmaceutically and therapeutically. Aim: The aim of the study is to carry out the stability of Bhallatakarishta (BA) prepared by two different liquid media with respect to its stability against microbial contamination. Methods: Both these BA samples were prepared and studied to check for microbial contamination at regular intervals. Results: Each time, the sample was subjected in microbial study from the day of preparation to the date of the last microbial study. No contamination was found in a microbial study in water media. In the one batch of Kanji (fermented sour gruel) media out of two batches, microbes were found after the 5th month date of preparations. Discussion: A current study was conducted to observe the stability of BA regarding microbial contamination for sample preparation and storage under different climatic conditions and temperatures. Thus, the basic microbial profile was studied at regular intervals for the consumption of the prepared drug. At the end of the study, it was found that the samples were not shown for the presence of any microorganisms in most of the collections. Conclusion: During the study period, no microorganisms were isolated from aerobic culture, and no fungal pathogens were identified from fungal culture in any batches of water media. Thus, these data support the long shelf-life of AsavaArishta formulation.

Keywords: Bhallatakarishta, Microbial Study, shelf-life


How to cite this article:
Bopaliya D, Parekh D, Cholera M, Patgiri B. Microbial study of Bhallatakarishta formulate by two different liquid media. J Ayurveda 2022;16:306-11

How to cite this URL:
Bopaliya D, Parekh D, Cholera M, Patgiri B. Microbial study of Bhallatakarishta formulate by two different liquid media. J Ayurveda [serial online] 2022 [cited 2023 Feb 6];16:306-11. Available from: http://www.journayu.in/text.asp?2022/16/4/306/364046




  Introduction Top


Asava and Arishta (fermented formulations) are medicinal preparations prepared by soaking medicines, either in the coarse powder form or in Kashaya (decoction) form, in a solution of sugar or jaggery, as the case may be, for a specified period of time during which they undergo a fermentation process to generate alcohol, which facilitates the extraction of the active substances contained in the medicines.[1] According to the Gazette of India, the shelf-life of AsavaArishta formulations is 10 years.[2] Classics of Ayurveda mentioned that Asava–Arishta becomes more and more effective as they become old.[3] However, one research article quoted that it may not be possible to have an unlimited shelf-life of any formulation. In AsavaArishta, self-generated alcohol may work as a preservative agent and it may reason for comparatively longer shelf-life.[4] First, Bhallatakarishta (BA) is explained in Ashtanga Samgraha Chikitsa Sthana Shotha Rogadhikara. A formulation composition of BA is given in [Table 1]. BA is a unique formulation from Asava–Arishta Kalpana due to its containing Mastu (whey) which is the byproduct of milk. Kanji provided acidic media for the extraction of chemical constituents of Kwatha Dravyas. In preparation of Kanji, fermentation process was happened. Hence, in this formulation, two times, the fermentation was taken place. Water is used as a solvent in almost the preparation of AsavaArishta. However, here, Kanji is given in this formulation maybe it is a better solvent for the drugs of BA. Hence, here, an attempt has been carried out to prepare Bhallatakarishta by Kanji (BAK) and water (BAW) as a liquid media. In the present study, two samples of BA were studied to test for microbiological contamination in the final product at diverse time durations under various environmental variables, temperatures, and humidity sets.
Table 1: Formulation composition of Bhallatakarishta

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  Materials and Methods Top


The study has obtained institutional ethics committee (PGT/7/-A/Ethics/2018-19/2638, dated December 18, 2018).

Procurement of drug

All the Kwatha Dravyas used for preparation of BA was procured from the Pharmacy, GAU, Jamnagar. Rice, Amul curd, sugar, and water were purchased from local market of Jamnagar. Authentication of all drugs was done through expert of Pharmacognosy Laboratory of IPGT and RA, GAU, and Jamnagar.

Drug preparation

Bhallataka Shodhana[5] and Chitraka Shodhana[6] were carried out as per the reference of Rasatarangini. Sour gruel was prepared as per the reference of Dravyaguna vigyaniyam.[7] Whey was prepared as per the reference of Raj Nighantu.[8] First, Kwatha (decoction) was prepared in water or Kanji as a liquid media with prescribed Kwatha Dravyas. After that, syrup of whey and sugar was prepared. Decoction and sugar syrup were mixed well and wort was prepared. The prepared wort was then put into a thoroughly cleaned, dry-fermented jar, filling 3/4 of the vessel's top space and closing the lid properly. After labeling, the vessel was placed in a clean, dry, and thoroughly fumigated sterile chamber to protect it from direct sunlight, air, and temperature changes. After receiving positive fermentation completion indicators, the supernatant liquid was strained through a double-folded cotton cloth and decanted into another properly clean, dried porcelain jar. In these protocols, two batches (BAW – A and B, BAK – A and B) of both the samples were prepared [Table 2].
Table 2: Data of different batches: fermentation initiation and completion


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BA was kept at room temperature and studied for microbial contamination from time to time. The biological reading has been conceded out in the Microbiology Laboratory, IPGT and RA, GAU, Jamnagar.

Storage

The finished products were stored in air-tight China clay jar containers in the department at room temperature.

Microbial profile

To check mycological and bacteriological findings, microbial contaminations have been assessed by two methods.

Smear examination

Wet mount/10% KOH preparation

Aim

To find out any mycological consequences.

Preparation of 10% KOH preparation

10% KOH solution is prepared by adding 10 g of KOH crystals to 50 ml distilled water. Mix until the crystals entirely dissolve. Add enduring distilled water to make the volume 100 ml. It is corrosive and should be handled with maintenance and stored at room temperature.

Sample preparation

A sanitary grease-free slide was taken. After that, a test drug droplet (i.e., BAK or BAW) was kept on the slide, and a freshly 10% KOH drop was added to it. After that, it was enclosed with a grease-free cover slip. Then, it was permitted to respond for 15–20 min to clear out any extra debris other than fungus. After that, the slide was observed under high power (×40) lens and observations were noted down properly.

Gram stain test

Aim

to rule out the phenotypic characterization of bacteria (Gram-positive or Gram-negative).

Sample preparation

  1. Smear preparation


  2. Smear preparation is required for many laboratory procedures, including Gram stain. The purpose of the smear is to fix the bacteria on the slide and prevent loss of the sample during the staining procedure. Place 1–2 drops of liquid material at the center of clean slide. The specimen was spread properly on the slide and slide was fixed by passing three to four times over the flame of Bunsen burner [Figure 1].
    Figure 1: Stained smear ready for examination

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  3. Staining procedure


It was done as per Gephardt et al. 1981, Feedback from ASMCUE participants, ASMCUE 2005.[9]

  1. Air-flood drying, heat-fixed swab cells for 1 min with crystal violet staining reagent. The quality of the smear will affect the result of the Gram stain
  2. Wash the slide in a gentle, indirect stream of tap water for 2 s
  3. Flood slide with mordant: gram iodine solution. Wait 1 min
  4. Wash the slide in a gentle, indirect stream of tap water for 2 s
  5. Flood slide with a decolorizing agent, i.e., acetone. Wait 15 s or drop to slide until the decolorizing agent is removed from the slide
  6. Flood slide with counter stain, safranin. Wait 30 s to 1 min
  7. Wash the slide in a gentle, indirect stream of tap water until no color appears in the drain and then dry it with absorbent paper
  8. Observe the results of the staining procedure under oil immersion using a bright-field microscope
  9. When the Gram stain is done, the Gram-negative bacteria will stain pink/red and will notice the Gram-positive bacteria in blue/purple color [Figure 1].


Culture study

Fungal culture

The culture method is the gold standard for the diagnosis of fungal infection. A culture has the advantage of producing the causative agent if it is positive. Furthermore, culture allows for sensitivity testing.

Sample collection

Test drug was collected by sterile cotton swab. Proper labeling was done, i.e., sample name, date of sample preparation, date of sample given, and storage condition – room temperature.

Prerequisites

Sabouraud dextrose agar (SDA) base (modified: dextrose agar base, Emmons purchased from HIMEDIA laboratories Pvt. Ltd.) [Figure 2], glass bottle, and incubator.
Figure 2: Fungal culture media

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Procedure

Before inoculating the specimens, the selective solid media was dried in a hot air oven. SDA base was filled in a small glass bottle which is named as SDA base bottle. The test sample was inoculated in the SDA base bottle by a sterile cotton swab. Incubate infected media in an inverted posture at 37°C for 5–21 days in an incubator under an aerobic atmosphere after the inoculation/streaking process. After that, bottle was removed from the incubator and examined by naked eyes in the form of a colony. After that, the report was generated.

Aerobic culture method

Sample collection

Cotton swab.

Aim

To isolate desired colonies from contaminates or sample.

Prerequisites

sterilized wire loop, Bunsen burner, MacConkey agar, and Coulmbia Blood agar (Hi Media Laboratories Pvt Ltd.), glass plates, and incubator.

Procedure (Streak plate method)

The procedure starts with wire loop being sterilized in a flame. Allow the sterilized loop to cool for a few seconds before using. Dip the loop into a test sample. Depending on the sample loop, hundreds or thousands of bacteria may be picked up and dispersed throughout the agar's surface. To avoid contamination, keep the dish covered as much as possible. Make a uniform smear with the inoculum at the edge of the plate. Sterilize the loop as mentioned above and make streaks from the smear. Since there is thinning of inoculum during streaking, isolated colonies are formed at the ideal streak point where the spreading has separated the individual (or group) of cells at locations where they can form individual colonies. During the incubation, the bacteria multiply and produce colonies. After the streaking process, incubate inoculated medium in an inverted position at 37°C for 24–48 h in the incubator under aerobic or 10% CO2 atmosphere. During streaking, intermittent burning of the loop can be done to lessen the load of an organism so that isolated colonies are less likely. To avoid contamination, keep the dish covered as much as possible. Try to keep the inoculating loop in touch with the agar surface at all times when streaking an area of the plate. Thereafter, the physical growth in the form of colon or aerial growth was assessed. After that, the report was isolated [Figure 3] and [Figure 4].
Figure 3: Aerobic culture media (MA)

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Figure 4: Aerobic culture media (BA)

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  Observations and Results Top


Observations and results of all the methods used for microbial examination of both the samples and both the batches are assumed in [Table 3] and [Table 4].
Table 3: Observations of Bhallatakarishta prepared by Kanji as a liquid media preserved at room temperature (Batch A, B)

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Table 4: Observations of Bhallatakarishta prepared by water as a liquid media preserved at room temperature (Batch A, B)

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  Discussion Top


The therapeutic regimen of Ayurveda has different categories of dosage forms of the drug, depending on the consistency and shelf-life, i.e., Swarasa (juice), Kwatha (decoction), Hima (cold infusion) and Phanta (hot infusion), Asava and Arishta (alcohol-containing preparation), Ghrita preparation or medicinal oil as liquid dosage forms, and Vati (tablets) and Churna (powder) as solid dosage forms, while semi-solid dosage preparation includes Kalka (paste) and Avaleha (confection).[10] There are different objectives of microbiology study so as to ensure any drug is safe to consume, to understand the role of climatic conditions in the growth of microbes. The growth of microbes also depends upon various factors such as storage condition and components of the formulation.

BA was prepared as per reference of Ashtanga Samgraha. The name of formulation is BA, but all the ingredients are observed in same proportion for preparation of decoction. Bhallataka is mentioned in Upavisha varga by Rasatarangini.[11] Bhallataka is also included in Schedule E[1] in Drug and Cosmetic Act.[12] Consequently, purification of Bhallataka is carried out before using therapeutically to avoid its toxic effect on the body.[13] The ingredients of the formulation are predominant with Katu (pungent) Rasa (taste), Laghu (lightness), Teekshna (sharpness) Ruksha (dryness), Guna (properties), Ushna (hotness), Veerya, Vipaka, and Kaphavatahara properties. Most of the drugs have Kushthghna (anti-dermatosis), Krimighna (anthelmintic), Kandughna (anthelmintic), and Rasayana (rejuvenation) properties. Whey is good source of carbon for the production of alcohol, single cell protein, Vitamin B12, lactic acid, and gibberellic acid.[14]

The medicine should be free of any microbial contamination for improved safety and efficacy. Microbial contamination should be avoided at all costs to ensure medicine stability and shelf-life. Both BA samples were prepared and stored at room temperature. During the study, temperature changes and humidity were observed.

In the present study, the main aim of microbiology is to ensure the safety and efficacy of both drug up to the consumption period of patient. After the 5th month of preparation, an Aspergillus niger microbe was revealed in aerobic culture in one batch of BAK media. The BAK-A batch was discarding after the detections of microbes. There is no microorganism isolated from aerobic culture and no fungal pathogens were isolated from fungal culture in the second batch of BAK [Table 3]. A microbe in BAK-A batch was found may be due to occurrence of any contaminations during the preparation and environmental factors such as humidity, temperature, light, storage condition, and packaging material. No microbes were isolated from any batches of BAW during the study period as a result of aerobic culture, and no fungal pathogen was isolated from any batches of BAW as a result of fungal culture [Table 4]. As the result of this study, researcher can reached to the conclusion that the environment and storage plays an important role for Asava-Arishta preparation. For more conformation of the stability of the formulation, one can go for stability study.


  Conclusion Top


The current study concluded that there is no growth in microorganisms in BA prepared by water as a liquid media. While one batch of Bhallatakrishta prepared by Kanji as a liquid media; shown the fungal growth after 5 months. For further confirmation of stability study of these two formulations, more batches can be evaluated or real-time stability study can be done.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.





 
  References Top

1.
The Ayurvedic Pharmacopoeia of India, Reprinted editions 1st, Govt. of India: Ministry of Health and Family Welfare; Monographs Part II: 2009.  Back to cited text no. 1
    
2.
CCRAS, General Guidelines For Drug Development Of Ayurvedic Formulations. Central Council for research in ayurvedic sciences Ministry of AYUSH, Government of India New Delhi. p.17.  Back to cited text no. 2
    
3.
Adhamala, Shastri K. Sharangdhara Samhita of Sharangdharachrya, Chukhamba Orientalia. Varanasi, Purva Khanda; Ch. 1 verse 53. 2016. p.13.  Back to cited text no. 3
    
4.
Gupta A, Jaiswal M, Prajapati PK. Shelf Life of Ayurveda dosage forms- Traditional view, current status, and prospective need. IJTK. 2011;10:672-7.  Back to cited text no. 4
    
5.
Shashtri K. Rasatarangini of Shadanand Sharma. 11th ed, Motilal Banarasidas Publication, Varanasi, ch. 24 verse 477-478. 1979. p. 735.  Back to cited text no. 5
    
6.
Shashtri K.Rasatarangini of Shadanand Sharma. 11th ed, Motilal Banarasidas Publication, Varanasi, ch. 24 verse 575. 1979. p. 753.  Back to cited text no. 6
    
7.
Vaidha Yadavji Trikamaji Acharya of Dravyaguna Vigyana, Niryasagar press, Bombay, Utaradha 1 pribhashakhanda, ver. 81. 2000. p. 41.  Back to cited text no. 7
    
8.
Raj Nighantu, Ksheeradivarga, 15/7. Available from: http://niimh.nic.in/ebooks/ enighantu. [Last accessed on 2021 Aug 05].  Back to cited text no. 8
    
9.
Smith AC, Hussey MA. Gram stains protocols. American society for microbiology 2016.  Back to cited text no. 9
    
10.
National Health Portal, Gateway of authentic health information. Publications: NHP Admin, Oct 24: 2021. https://www.nhp.gov.in/processing-in-ayurveda_mtl.  Back to cited text no. 10
    
11.
Shashtri K, Rasatarangini of Shadanand Sharma, 11th ed, Motilal Banarasidas Publication, Varanasi, ch. 24 verse 163. 1979. p. 676.  Back to cited text no. 11
    
12.
Deshpande SW, Gandhi N. Drug and cosmetic act 1940 and rules 1945, 9th ed. susmit publishers; 2018. p. 463.  Back to cited text no. 12
    
13.
Pawade Udey Venkatrao. A toxicological review of Bhallataka (Semicarpus anacardium Linn. IAMJ; 2015:3:2477-87.  Back to cited text no. 13
    
14.
Satyanarayan U. Biotechnology. Bioprocess, fermentation technology 12th ed. Uppala Auther Publisher interlink, ch. 19. 2017. p. 249.  Back to cited text no. 14
    


    Figures

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

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



 

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