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| ORIGINAL ARTICLE |
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| Year : 2022 | Volume
: 7
| Issue : 1 | Page : 23-37 |
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Pharmaceutical standardization of Nagaphani Phalasava (fermented dosage form of Opuntia elatior Mill. fruits)
Sharada Anand, Rabinarayan Acharya
Department of Dravyaguna, ITRA, Gujarat Ayurved University, Jamnagar, Gujarat, India
| Date of Submission | 16-Dec-2021 |
| Date of Decision | 03-Feb-2022 |
| Date of Acceptance | 03-Feb-2022 |
| Date of Web Publication | 07-Jul-2022 |
Correspondence Address:
Dr. Sharada Anand
Department of Dravyaguna, ITRA, Pandit Nehru Marg, Swastik Society, Park Colony, Jamnagar 361008, Gujarat
India
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/jdras.jdras_81_21
BACKGROUND: Fruits of Opuntia elatior Mill. (OE) Cactaceae, commonly recognized as Red Prickly pear, is reported for multiple pharmacological actions, yet under-utilized due to its perishable nature and seasonal availability. Asava not only helps in elongation of shelf life but also offers continued extraction of the active compounds. Hence, the study has been carried out to develop and standardize a novel dosage form Nagaphani Phalasava (fermented preparation of OE fruits). MATERIALS AND METHODS: Asava was prepared in two seasons, that is, winter (December 2020–January 2021) and summer (April 2021–May 2021). A total of nine pilot batches were prepared in each season, that is, 7, 10, 12, 15, 18, 21, 24, 27, and 30 days, considering the duration of fermentation. Each batch was opened the next day, that is, 8th, 11th, 13th, 16th, 19th, 22nd, 25th, 28th, and 31st day, respectively, to observe the tests of perfectness and carry out physicochemical analysis including pH, specific gravity (Sp gr), density, total solids, alcohol percentage, and sugar analysis. The batch that showed all the tests of perfectness was considered ideal and repeated in triplicate. RESULTS: In both seasons, 30 days batch was considered ideal and repeated in triplicate. Yield was 68.73% in winter and 59.44% in summer season. Organoleptic features were more prominent in winter batch compared to summer batch. Average pH, Sp gr, density, total solids, alcohol percentage, total sugar, reducing sugar, and nonreducing sugar were 2, 1.0591, 1.0225, 20.48, 11, 17.56, 4.6, and 12.9 in winter and 2, 1.1198, 1.0707, 27.31, 4, 12.3, 7.5, and 4.8 in summer, respectively. CONCLUSION: Preparation of Nagaphani phalasava requires a duration of 30 days in both winter and summer seasons. The winter batch Asava possessed more evident organoleptic features compared to summer batch. Product yield was comparatively high in winter. The alcohol percentage was under the limits prescribed for Asavarishta in Ayurvedic Pharmacopoeia of India (API). Keywords: Dosage form, Nagaphani phalasava, Opuntia elatior, standardization
How to cite this article:
Anand S, Acharya R. Pharmaceutical standardization of Nagaphani Phalasava (fermented dosage form of Opuntia elatior Mill. fruits). J Drug Res Ayurvedic Sci 2022;7:23-37 |
How to cite this URL:
Anand S, Acharya R. Pharmaceutical standardization of Nagaphani Phalasava (fermented dosage form of Opuntia elatior Mill. fruits). J Drug Res Ayurvedic Sci [serial online] 2022 [cited 2023 Sep 23];7:23-37. Available from: http://www.jdrasccras.com/text.asp?2022/7/1/23/350058 |
| Introduction | |  |
Pharmaceutical science in Ayurveda started with five basic dosage forms, that is, Svarasa (fresh Juice), Kalka (paste), Kwatha (decoction), Hima (cold water infusion), and Phanta (hot water infusion).[1] Eventually, various other dosage forms were developed, in view of increased shelf life, palatability, and bioavailability.[2]
Ayurvedic Formulary of India (AFI) has explained Asava, Arishta, Arka, Avaleha, Guggulu, Ghrita, Churna, Taila, Lepa, and Vati dosage forms with suitable examples. This compendium mentions 57 Asavaristas in Parts I (37), II (3), and III (17)[3],[4],[5]
These are the fermented preparations that provide a long shelf-life and help in the continued extraction of the active compounds of the drug. Different parts of the plant are used in the preparation of Asava (Asavayoni), such as Dhanya (grain), Phala (fruit), Mula (root), Sara (exudate), Pushpa (flower) Kanda (stem), Patra (leaf), and Twak (bark). Fruit, the most quickly perishable part of a plant, is also a Asavayoni. A total of 26 Phalasava are mentioned in the classics.[6]Drakshasava,[1],[7],[8]Kharjurasava,[7],[8] and Pilvasava[9] are some of the classical phalasavas prepared using the pulp of the fruit. Difference of opinion regarding the duration of fermentation from 7 days to 180 days have been recorded.[10],[11]
Nagaphani (Opuntia elatior Mill. [OE]), Cactaceae, is one such ethno-medicinally important plant wherein fruit is the major part used as a nutritional supplement and medicine,[12] yet they are under-utilized.[13] It is reported to be rich in carbohydrates, flavonoids, phenolics, betalains, and vitamin C and for various activities like anti-oxidant, hematinic, anti-leukemic, antidiabetic, analgesic, and anti-inflammatory. It is safe for administration in a dose-dependent manner.[12]
Fruit as such or fruit juice is commonly consumed, which has a short shelf life. Few dosage forms such as juice[14] and jelly[15] have been formulated and are available in the market.
A dosage form should fulfill the basic requirements of being safe and effective, for which, standardization is the pivotal step that assures quality, efficacy, safety, and reproducibility.[16] Hence, this study has been carried out to develop and standardize a novel dosage form Nagaphani Phalasava (fermented preparation from Opuntia elatior fruits), considering season and duration of fermentation.
| Materials and Methods | | |
The study has been carried out in the Departmental and Pharmaceutical laboratory, ITRA, Jamnagar. Nagaphani Phalasava was prepared in two seasons, that is, winter (December–Jan 2020) and summer (April–May 2021) considering the seasonal availability of fruits.
Procurement of raw drugs
Nagaphani phala (OE fruits) were collected from peripheral areas of Jamnagar, Gujarat, following good collection practice guidelines,[17] after confirming its identity by taxonomist as OE, considering the morphological characters. Sample specimen was authenticated by the expert of pharmacognosy laboratory of Gujarat Ayurved University, Jamnagar and samples were deposited to Institutes pharmacognosy museum [IPGTandRA/PHM-6344/20–21] for future references.
Sugarcane-based Guda (jaggery, brand- Rajbhog natural) and Dhataki pushpa (Woodfordia fruticose (L.) Kurz. flowers) were procured from a local vendor after confirming the identity and purity. Dhoopana dravya (drugs for fumigation), that is, Guggulu (Commiphora wightii (Arn.) Bhandari.), Vacha (Acorus calamus L.), and Haridra (Curcuma longa L.) were collected from institutional pharmacy, after proper authentication. All the equipment and chemicals used for the analytical study were in accordance with the Ayurvedic Pharmacopoeia of India (API) guidelines.[18]
The equipment specifications are detailed in [Table 1].
The ingredients used for the preparation with quantity are depicted in [Table 2].
Methods
Principle
Preparation was carried out based on the Asava nirmana vidhi (methods of preparing fermented dosage form) explained in the classical literatures of Ayurveda.[1] As OE is an extra pharmacopoeial drug, the fixation of proportion of ingredients and duration of fermentation was done after a thorough investigation of the classical and compiled books with regards to Asava of pulpy fruits[1],[7],[8],[9],[19] and few scientific review articles.[20],[21] A duration between 7 days and 30 days of fermentation was selected here for the study, considering usual range of duration told for the fermentation of Asavarishtha in the classical literatures of Ayurveda.[22]
Preparation of Svarasa
Fully riped OE fruits were collected adopting few precautionary measures for protection from thorns like use of hand gloves and long-armed tongs to pluck the fruits. After removing the thorns, fruits were washed under running tap water to remove adherent dirt. Cleaned fruits were peeled off to expose the inner pulp. While peeling, care was taken to remove the thin spines (glochids) attached to the outer skin of the fruit. The pulp was ground well to extract the juice. This juice was then filtered through stainless steel strainer of mesh size 0.0625 inches. The seeds were discarded. Thick and frothy svarasa thus obtained was used for preparation of Asava [Figure 1].
Preparation of Asava
The containers were pre-cleaned with hot water and dried in sunlight and subjected to Dhupana using Dhupana Dravya. After Dhupana the lid was closed and the containers were kept undisturbed for 24 h. A total of nine pilot batches were prepared, in each season, considering the duration of fermentation (7, 10, 12, 15, 18, 21, 24, 27, and 30 days). Among them, the batch which showed all the signs of completion of fermentation was considered ideal and repeated in triplicate. The svarasa was mixed with guda (organic jaggery) in the specified proportion. The mixture was stirred well until guda gets completely mixed in the svarasa. The solution containing svarasa and guda was filtered and filled into the containers leaving half portion of the container empty. Dried Dhataki pushpa churna was mixed to this solution at the end. Containers were closed using the lid. A day later, sandhibandhana (sealing the containers) was performed using cotton cloth (10 cm wide and 200 cm long) smeared with Multani mitti (Fuller earth).
Each sealed container was labeled consecutively like 7, 10, 12, 15, 18, 21, 24, 27, and 30 days batch and kept for fermentation. Room thermometer (brand: Gera plastic wet and dry bulb) was hung on the wall near the containers to record the temperature on daily basis [Figure 1].
Precaution
All the utensils used in the process were sterilized by washing with hot water and heating. All the containers were subjected to dhupana karma using the dhupana dravya mentioned earlier. After dhupana, the lids were closed and the containers were kept aside for a period of 24 h. Proper sanitation was maintained during the pharmaceutical procedure. Adequate space was left in the Sandhana Patra for the circulation of the liberated gas, during fermentation. The fermenting vessels were kept in places devoid of direct sunlight, air, and temperature variations. Filtering was done by using a starch-free, clean, dry cotton cloth.
Assessing for tests of completion of fermentation
After completion of the scheduled duration of all the nine pilot batches as explained above, containers were opened the next day, that is, 8th, 11th, 13th, 16th, 19th, 22nd, 25th, 28th, and 31st day respectively, and observed for tests of completion of fermentation perfectness of fermentation, which included burning matchstick test, presence of effervescence, floating particle and alcoholic odor[20] [Figure 2]. | Figure 2: Depicting assessment of tests of completion of fermentation, product filtration, and storage
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Final product
After fermentation, the solution was carefully filtered using unstarched muslin cloth, the yield was recorded and the final product (Asava) was transferred into previously fumigated glass container. The containers were sealed properly and labeled with all the necessary information. The same procedure was adopted for all the batches prepared.
Analytical study
In each batch, after observing for the tests of completion of fermentation, sufficient quantity, that is, 170 mL of the filtrate was taken for carrying out organoleptic and physicochemical analysis. Organoleptic evaluation was done with the help of healthy volunteers based on the special proforma designed for the purpose. After taking the filtrate, the containers were sealed back and kept for fermentation for 30 days to look for any further changes in organoleptic and physicochemical parameters.
Analytical tests mentioned for Asava, that is, pH, Specific gravity, Density, Viscosity, Total solids, Alcohol percentage, Sugars were performed following the API guidelines.[18] Methanolic extract of Asava (the total solid content obtained after boiling on water bath, was macerated with 5 ml methanol) of all the batches of both the seasons was evaluated for high-performance thin-layer chromatography (HPTLC) profiling using Toluene and ethyl acetate in the ratio 9:1, as mobile phase. Scanning was done under ultraviolet (UV) (254 nm) and UV (366 nm).
All the methods adopted in the study are schematically represented in [Figure 3].
| Observations and Results | | |
Collection and processing
Availability of well-ripened fruits was observed in both winter (mid-November to January) and summer season (April to June). Collection of the fruits was difficult due to the presence of thorns and glochids on the fruits. Fruits were highly perishable in nature.
Preparation of svarasa
The prepared svarasa was thick and frothy in consistency. Hence it was difficult to filter through muslin cloth. Therefore, a stainless steel strainer with mesh number 0.0625 inch was used. Percentage yield of svarasa was noted to be 74.9% in winter and 62.6% in summer.
Temperature range
During the winter season, the temperature range was recorded to be from 21ºC to 25°C and in summer it ranged from 30°C to 35°C.
Tests for completion of fermentation
In winter, the burning match stick test was positive (continued to burn when taken near the fluid) as early as 12 days of fermentation whereas, in summer, the test was positive as early as 10 days. Effervescence/hissing sound started to reduce from 18 days batch in winter and 24 days batch in summer. Floating particles started to disappear and the solution became clear gradually from 18 days batch in winter and 24 days batch in summer. In winter, the alcoholic odor was witnessed from 15 days batch but in summer, it was witnessed from 12 days batch. In the final batch, all 3 batches showed positive results in all the tests of completion of fermentation. The solution was very clear and transparent, there was no hissing sound heard and a very strong alcoholic odor was witnessed as soon as the container was opened. Consistency of Asava was thinner in winter compared to the summer batch.
Organoleptic characters
In both the seasons, color of the solution turned from dark mulberry to dark raspberry by the end of 30 days. Characteristic sweet odor of the solution changed to strong alcoholic odor by the completion of fermentation. Odor was stronger in the winter batch compared to the summer batch. Taste changed from Madhura-amla rasa (sweet-sour) to Madhura-Amla-Kashaya (sweet-sour-astringent) after completion of fermentation. Ushnata (hot sensation) was felt at the throat on intake. Consistency became thin as the fermentation proceeded. The final product was very thin, clear, and translucent. Asava of the winter season was comparatively thinner and clearer than the summer season.
Yield
The average yield was noted to be 63.23% and 68.73% in pilot batches and final batches of the winter season, respectively, whereas, in the summer season, the average yield was found to be 59.83% and 59.44% in pilot and final batches, respectively.
Physicochemical parameters
The physicochemical parameters of winter pilot batches are shown in [Table 3]. | Table 3: Results of physicochemical analysis of winter season pilot batches
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The physicochemical parameters of winter final batches are shown in [Table 4]. | Table 4: Results of physicochemical analysis of winter season final batches
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The physicochemical parameters of summer pilot batches are shown in [Table 5]. | Table 5: Results of physicochemical analysis of summer season pilot batches
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The physicochemical parameters of summer final batches are shown in [Table 6]. | Table 6: Results of physicochemical analysis of summer season final batches
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The comparative results of the physicochemical analysis are depicted in [Graph 1][Graph 2][Graph 3][Graph 4][Graph 5][Graph 6][Graph 7][Graph 8].
HPTLC profile
The results of the HPTLC study are presented in [Table 7] and [Figure 4][Figure 5][Figure 6][Figure 7]. | Table 7: Results of high performance thin layer chromatography of Nagaphani phalasava
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 | Figure 4: Depicting HPTLC profile of alcoholic extract of all batches of Nagaphani Asava of winter season at 254 nm: (A) 7 days batch, (B) 10 days batch, (C) 12 days batch, (D) 15 days batch, (E) 18 days batch, (F) 21 days batch, (G) 24 days batch, (H) 27 days batch, and (I) 30 days batch
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 | Figure 5: Depicting HPTLC profile of alcoholic extract of all batches of Nagaphani Asava of winter season at 366 nm: (A) 7 days batch, (B) 10 days batch, (C) 12 days batch, (D) 15 days batch, (E) 18 days batch, (F) 21 days batch, (G) 24 days batch, (H) 27 days batch, and (I) 30 days batch
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 | Figure 6: Depicting HPTLC profile of alcoholic extract of all batches of Nagaphani Asava of summer season at 254 nm: (A) 7 days batch, (B) 10 days batch, (C) 12 days batch, (D) 15 days batch, (E) 18 days batch, (F) 21 days batch, (G) 24 days batch, (H) 27 days batch, and (I) 30 days batch
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 | Figure 7: Depicting HPTLC profile of alcoholic extract of all batches of Nagaphani Asava of summer season at 366 nm: (A) 7 days batch, (B) 10 days batch, (C) 12 days batch, (D) 15 days batch, (E) 18 days batch, (F) 21 days batch, (G) 24 days batch, (H) 27 days batch, and (I) 30 days batch
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| Discussion | | |
Asavarista is considered as a fermented dosage form in which the microbial activity initiates alcohol formation rendering increased extraction of active constituents and amplified bioavailability.[22],[23] The dosage form is well established in the classical literatures of Ayurveda since ages. Biotransformation in this dosage form is interceded by the microorganisms, which also help in its preservation. These preparations are more appreciated among all the formulations due to their quick action and longer shelf life.[21],[24]
Duration of fermentation ranges from 7 days to 180 days in different Asava formulation.[10],[11] Duration of fermentation varies due to many attributable factors, the season being one among them. Literature reveals, during autumn and summer seasons it takes 6 days for fermentation, 10 days in winter, and 8 days in rainy and spring seasons.[20]
Sweetening agent, jaggery was added to aid continued fermentation and prevent putrefaction of the fluid. The quantity of jaggery was calculated considering the classical literatures[1],[19] and scientific articles.[24] In this study, the temperature range was recorded from 21°C to 25°C in winter and 30°C–36°C. Ideal duration of fermentation was seen to be 30 days in both winter and summer seasons. Though initiation of fermentation was fast in summer compared to the winter season, complete fermentation was achieved only after 30 days in both seasons. The final product in winter was thinner and clear fluid compared to that in the summer season. There were no signs of contamination or fungal growth seen on the product throughout the procedure which reassured that the procedures followed for sterilization were satisfactory. The pH was more acidic in the summer season than in winter. In winter, the specific gravity and density decreased gradually as the duration of fermentation increased whereas in summer this pattern was not evident. This can be attributed to the presence of less dense particles due to the warmth in the solution, which decreases the sedimentation rate thereby hindering the activity of micro-organisms and fermentation process.[20] Process of fermentation in sandhana kalpana is majorly performed by yeast. In ancient times fermentation was mainly initiated by the addition of Dhataki flowers (Woodfordia fruticosa Kurz.) which act as a source of yeast for fermentation.[22] A total of 24 yeast strains were isolated from the flowers of W. fruticosa that show the diversity of micro-organisms utilized in fermentation. Micro-organisms responsible for fermentation differ variably owing to the yeast and sugar source used and variation in temperature.[21],[23],[24],[26] Microorganisms involved in the fermentation could be diverse in both seasons, which might be the reason for the difference in results. The pH ranged from 4-2, indicating the presence of strong acids such as sulfuric, nitric, and carbonic acids which further help in the preservation of the product and prevent the growth of bacteria and other harmful microbes that speed up the decay process.[25],[27] pH is the main factor affecting the stability of vitamin C; thus, high values of pH favor the oxidation processes of vitamin C.[28] Sugar present as a sweetening substance is reduced and converted to alcohol, which helps dissolution of the active principle into the liquid media.[20],[23],[27] Hence there is a gradual decrease in reducing sugar. The reduction in sugar may be due to its utilization by the non-alcohol fermenting organisms. The total dissolved solids content decreases gradually in the winter season, suggesting the conversion of solid entities into liquid and gaseous forms.[23],[27] It is evident that alcohol generation seems to start from Day 7 and the alcohol content reaches a 12% level by the 21st day. This is by the Indian pharmacopeia standards established and suggested for the percentage of alcohol present in Asavarishtas.[18] HPTLC is one of the ideal TLC techniques for analytical purposes because of its increased accuracy, reproducibility, and ability to document the results. In this study, alcoholic extract of Nagaphani Asava is studied for HPTLC profile at 254 nm and 366 nm frequency. HPTLC study showed the presence of a maximum of 7 spots in winter and 5 spots in summer at short UV and 8 spots in winter and 2 spots in summer at long UV. Lesser peaks were observed under 366 nm when compared to 254 nm in both the batches.
| Conclusion | | |
Preparation of Nagaphani phalasava requires a duration of 30 days in both winter and summer seasons. The prepared Asava possesses Madhura-Amla-Kashaya and ushna guna. Asava of the winter season was thinner and clearer in consistency, teekshna (sharp) in comparison with the summer season. Product yield was comparatively high in winter (68.73%). The alcohol percentage was higher in the winter season than summer and under the limits prescribed for Asavarishta in API. The drug Opuntia elatior is proven safe and non-toxic for internal administration and the quality standards of the Asava were in accordance with the API standards given for Asava Arishta. Hence the novel dosage form Nagaphani Asava can be assumed safe and nontoxic for use. Further works on microbial activity during fermentation process, quantitative estimation, heavy metal analysis, and experimental pharmacology can be carried out to better understand the comparative differences.
Financial support and sponsorship
ITRA, Jamnagar, Ministry of Ayush.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]
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