Essential Parameters for preparation of Ayurvedic Fermented Products Asava and Arista: An Overview

 

Chandan Das, Debajyoti Das*

School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan Deemed to be University,

Bhubaneswar 751003, Odisha, India

 

ABSTRACT:

Ayurveda, meaning the science of life, is one of the ancient systems of medicine which is being practiced for several thousands of years by Indian civilization. Ayurveda consists of different type of dosage forms among which asava and arista are stated as the fermented products as these are exposed to the fermentation by the addition of fermentative agents, Saccharomyces cerevisiae obtained from dhataki puspa. Asava and arista, the unique dosage forms of Ayurveda known as “older the better it is” due to their easy palatability, effectiveness and indefinite shelf life. Preparation of asava and arista was described under ‘Madyakalpana’ and were emphasised during Samhita period due to their faster absorption and longer shelf life. The use of these formulations has been proved during Vedic period by many researchers which were a revolutionary innovation during that period.  In this article attempt is made to describe the different constituents essential for preparation of asava and arista. These involve dravya (drug substances), dravadravya (solvents), sandhaneeya dravya (fermentative agents), prakshepa dravya (additives) and madhura dravya (sweetners). Further, sandhana patra (fermentation container), patra samskaras prior to sandhana (pretreatment of container before fermentation), sandhana sthala (fermentation place), sandhana avadhi (duration of fermentation), time of fermentation and the duration of process for preparing asava and arista have also been highlighted.

 

 

 

INTRODUCTION:

The history of health care goes back beyond 5000 years BC in India where diseases were recorded in ancient texts like ‘Rig Veda’ and ‘Atharva Veda’. Later, the texts like ‘Charak Samhita’ and ‘Sushruta Samhita’ were documented in about 1000 years BC. In recent time standardization parameters of plant derived products have been described in modern texts such as official compendias like Ayurvedic Pharmacopoeia of India and Ayurvedic Formulary of India. Ayurveda is one of the oldest systems of medicine, the concepts and approaches of which have been perfected between 2500 and 500 BC in ancient Indian subcontinent^1,2.

 

 

 

Ayurvedic formulations are the mixture of multi-component drugs, which consist of products derived from plants, animals, minerals and metals. Ayurveda include several formulations like Arista, Asava, Churna, Ghrita, Gutika, Vati, Taila, Avaleha, Lepa and Praash. Apart from these Ayurveda also include some other formulations such as Lauha, Arka, Kvatha, Dravaka, Lavana Ksara, Guggulu, Rasa-Yoga, Pisti, Parpati Kalpas, Sattava, Kupipakva and Rasayana, Vartti-Netrabindu and Anjana, Mandura, Kalka and Swarasa^3,4.

 

Among all these formulations, asava and arista, considered as alcoholic preparation, are popular over all other dosage forms due to their continuous hydro-alcoholic extraction of several phytoconstituents from crude drug into the medium. Self generated alcohol makes the product more potent by preserving them for longer duration⁵. This review aimed at providing the basic requirements for the preparation of asava and arista formulations. The sanskrit names of various ingredients as mentioned in the classic texts are used in the description.

METHODOLOGY:

Relevant literature was collected from ancient ayurvedic classics books such as Charaka Samhita, Sushruta Samhita, Ashtanga Hridaya, Ashtanga Sangraha, Sharangadhara Samhita, Gada Nigraha, Yogaratnakara, Bhaishajya Ratnavali, Pharmacopoeial standards for ayurvedic formulations, a manual of Indian Pharmacopoeia, Ayurvedic Pharmacopoeia of India and  Ayurvedic Formulary of India. Informations were also collected from electronic data base such as Elsevier-Science direct, Google scholar and Pubmed.

 

BASIC REQUIREMENTS OF ASAVA AND ARISTA PREPARATION:

Asava and arista are two ayurvedic medicinal formulations prepared by soaking the drugs either in powder form or kashaya (decoction) form in a solution of sugar or jaggery for a specific period of time to facilitate fermentation for generation of alcohol which is carried out by addition of dhataki pushpa (Woodfordia fruticosa). The self generated alcohol, thus obtained, act as preservative and facilitate the hydroalcoholic extraction of active principles present in the crude drug⁶. Preparation of asava and arista need some basic ingredients which include dravya (drug substances), drava dravya (solvents), sandhna dravya (fermentative agents), madhura dravya (sweetners) and prakshepa dravya (additives).

 

Dravya (Drug substances):

Dravyas are the basic ingredients of asava and arista formulation which are collected in required quantity after their authentification as per the standards mentioned in Ayurvedic Pharmacopoeia of India. Raw materials used for formulation should be free from foreign matter and other residual contaminants or pesticides⁷. The drugs used for preparation of asava and arista are obtained from three main sources, i.e., herbal, mineral and animal origin. Dhanya (cereals), moola (roots), phala (fruits), kanda (tuber), pushpa (flowers), patra (leaves), twak (stem bark), saara (heart wood), sarkara dravya (sweetening agents) are nine yonis (ingredients) advised by ‘Charaka’ for preparation of different asavaristas. Also, the use of bhasma (ash) of certain drugs (Palasa bhasma and Tilanala bhasma) and loha churna (iron powder) are recommended by ‘Susruta’ for this purpose. From animal source, ghee and madhu are generally used to anoint the vessel or pot to be used as the container for fermentation⁸. The dravya used in the preparation of arista are twak (bark), moola (root) which are kathina (hard) or madhyama dravyas (medium hard) and these are prepared into kashaya (decoction). The drug substances used in asava are generally mrudu (soft), volatile in nature viz; karpoora, chandana, ushira etc, which are made as hima (cold decoction)/phanta (hot decoction)/ swarasa (expressed juice)^{9, 10}.

 

Drava dravya (Solvents):

In asavarista formulation, medicinal plant parts are extracted by infusion and decoction methods. Different classic texts have mentioned about many liquids used as the solvent, out of which water is the most common one. Other liquids include plant juice, fruit juice, decoctions, butter milk, curd water, gomutra (cow urine), kanji and dhanyamla etc. It is also observed that as solvent two or three liquids are combined together in some formulations. Different drava dravyas used in different formulation are enlisted in (Table 1).

 

Table 1. Drava dravya generally used in asavarista

Sl. No	Drava dravya	Example	References
1	Jala (water)	Pippalyasava, Ushirasava	[11]
2	Gomutra (cows urine)	Chitrakasava	[11]
3	Dadhi (curd)	Gandeerasava	[12]
4	Gomayarasa (cowdung)	Gandeerasava	[12]
5	Takra (buttermilk)	Takrarishta	[13]
6	Kwatha (decoction)	Dasamoolarishta, Duralabhasava, Punarnavasava, Vasakasava	[14]
7	Swarasa (expressed juice)	Dhatryarishta, Dhatryarishta, Madhvarishta, Kumaryaasav, Vasarishta	[15]

 

 

Sandhana Dravya (Fermentative agents):

Dhataki pushpa (Woodfordia fruticosa), madhuca pushpa (Madhuca longifolia, Macbr), surabeeja (mother yeast), kinva (microbial inoculums), yeast and rarely puga, badara twak (bark), babbula twak (bark) serve as natural carriers of fermenting organisms which initiate fermentation process. Acharya Charaka has described the properties of dhataki pushpa but it was first used by Acharya Vagbhatta for fermentation for sandhana kalpana (fermented product)¹⁶. Madhuka pushpa and dhataki pushpa are mentioned in Sharangadhara Samhita and Ashtanga Hrudaya as Sandhana Dravya^17-19. Presence of tannins in these flowers favour the growth of yeast by providing suitable environment. Honey, resins and Madhuca flowers contain wild yeast which may be added to the preparation to initiate the fermentation process in absence of dhataki pushpa²⁰. These flowers are nectariferous and highly tanniferous, which contain yeast spores in the dry nectariferous region^21-23. Different fermentative agents used in preparation of asavarista formulations are given in (Table 2).

Table 2. Name of Sandhana dravya (Fermentative agents)

Sl. No	Sandhana dravya	Example	References
1	Dhataki pushpa	Abhayarishta, Arjunarishta, Aravindasava, Asokarista, Asvagandhadyarista, Usirasava, Kanakasava, Kutajarista, Kumaryasava, Khadirarista, Candanasava, Jirakadyarista, Dantyadyarista, Dasamularista, Devadarvarista, Draksarista, Parthadyarista, Pippalyadyasava, Punarnavasava, Balarista, Mustakarista, Mrdvikarista, Rohitakarista, Lohasava, Vasakasava, Vidangarista, Srikhandasava, Sarasvatarista, Sarivadyasava.	[14]
2	Madhuka pushpa	Ahiphenasava, Kutajarishta	[14]
3	Surabeeja (mother yeast) / Kinva (microbial inoculums)	Sura	[24]
4	Yeast	Arjunarishta	[25]
		Ashvagandharishta	[26]
		Drakshasava	[27]
		Draksharishta	[28]
		Kanakbindvarishta	[29]
		Nimba arishta	[30]

 

Surabeeja/Kinva:

Residue obtained after filtration of the final product of fermentation is called ‘surabeeja’ or ‘kinva’ which is dried and stored for further use. Chanakya has elaborated the process of preparation of kinva from raw and cooked grains in his famous text Kautilya Arthashastra. There, it is mentioned that same surabeeja should be used every time for maintaining the consistency in quality of an asava or arista. ‘Kinva’ which is obtained from different type of grains may alter the taste of the formulation¹⁷.

 

Use of Yeast:

Inoculums of yeast are used to initiate the process of fermentation. These are derived from the flowers which contain wild species of yeast. Enzymatic conversion to alcohol in anaerobic condition was brought by this flowers³¹.

 

Madhura dravya (Sweetening agents):

Madhura dravya is essential which provide the base for fermentation. Some madhura dravyas such as guda (jaggery), sharkara (sugar), phanita (molasses), sitopala (candy sugar), ‘Matsyandika’ & Khanda sita (different types of jaggary & sugar) have unique bio-chemical attributes which are used in specific quantities and act as source of carbohydrates in asavarista preparations. These sweet substances trigger and energize bacterial growth in the fermentation medium and causes fermentation of amalgamated mix. Rate of fermentation and quality of the final product depends upon the nature, quantity and quality of sweetening substances. The presence of molasses with alkaline content causes in faster fermentation where as honey may make the process slower but sustained^32-34. 40% sugar is the most ideal quantity for preparation of asavaristas. According to Acharya Sharangadhara, if Drava dravya is one Drona (12.288litre), the proportion of jagerry and honey should be one Tula (4.8kg) and half Tula respectively. If the above ratio becomes equal or doubles the quantity of Dravyas, it causes difficulty in dissolution of sweet substances and makes the whole mixture viscous and may causes less chance of fermentation. Growing of yeast is best observed in sugar solution of 30-40%, as they can tolerate high concentration of sugar³⁵. Various madhura dravyas used in different asavarista formulations are mentioned in (Table 3). Addition of minimum and maximum percentage of madhura dravya according to different texts is given in (Table 4).

 

 

Table 3. Madhura dravya used in preparation of asava and arista formulation

Sl. No	Madhura dravya	Example	References
1	Guda (Jaggery)	Kutajarishta, Abhayarishta, Amrtarishta, Ayaskrti, Arjunarishta, Asokarista, Kutajarista, Kumaryasava, Candanasava, Jirakadyarista, Dantyadyarista, Dasamularista, Draksarista, Parthadyarista, Pippalyadyasava, Balarista, Mustakarista, Rohitakarista, Lohasava, Vasakasava, Srikhandasava, Sarivadyasava.	[14]
2	Madhu (Honey)	Ayaskrti, Vidangarishta, Asvagandhadyarista, Usirasava, Kumaryasava, Khadirarista, Dasamularista, Devadaravarista, Punarnavasava, Madhukasava, Mrdvikarista, Rodhrasava, Vidangarista, Sarasvatarista.	[14]
3	Sharkara (sugar)	Ushirasava, Aravindasava, Kanakasava, Khadirarista, Candanasava.	[14]
4	Phanita (molasses)	Pippalyasava, Kutajarishta.	[36]
5	Madhu (Honey) and molasses	Usirasava	[14]
		Kumaryasava, Drakshasava	[15]
6	Madhu (Honey) and sharkara (sugar)	Drakshasava, Kumaryasava	[15]
		Usheerasava	[11]
		Mrudvikarishta	[37]

 

 

 

Table 4. Minimum and maximum percentage of sweetening agent

Sl. No	Name of Treatise	Minimum (%)	Maximum (%)	References
1	Charaka Samhita	15.18-20	150-156.25	[8,10]
2	Sushruta Samhita	25-39	178.57-400	[8, 10]
3	Ashtanga Hrudaya	20.23-39	156.25	[8, 10]
4	Astanga Hangraha	21.9	156.25	[8, 10]
5	Sharangadhara Samhita	32.03	156.25	[8, 10]

 

Prakshepa dravya (Additives):

Some of prakshepa dravyas include lavanga, ela, twakpatra, nagakesara, trikatu etc., which are added to the formulation after preparation of decoction. They impart aroma, colour and taste to the formulation and make it free from contamination due to their tikshana guna (antifungal, antiseptic, bactericidal properties). These are added in the form yavakuta (coarse powder) or fine powder to the sandhana patra. In some asavarista formulations, metals (Loha, Tamra, Swarna) are mentioned as ingredients. Thin gold leaves are added in Sarasvataristhta. Metals are not dissolved in Sandhana dravya but enhance the property of ingredients³⁵.

 

Different texts suggest the addition of fine powders of metals on different days such as 1st, 4th, 5th day during or after the completion of the fermentation. Sometime additives are added or spread over wort in the form of a paste and followed by homogeneous mixing in order to facilitate exchange of air or oxygen at the beginning of the fermentation and thereby restrain the growth of micro-organisms for proper fermentation^38-40. Use of Prakshepa dravyas in different formulation are mentioned in (Table 5).

 

Table 5. Prakshepa dravya used in different formulations

Sl. No	Name of Formulations	Name of Prakshepa dravya	References
1	Amrtarista	Sveta Jiraka, raktapuspaka, saptacchada (Saptaparna), sunthi, marica, pippali, nagakesara, musta, prativisa, indrayava.	[14]
2	Asokarista	Svetajiraka,musta, sunthi, daruharidra, utpala, haritaki, bibhitaka , amalaki, amra, vasa, sveta candana.	[14]
3	Asvagandhadyarista	Sunthi, marica, pippali, tvak, ela, tejapatra, priyangu, nagakesara.	[14]
4	Babbularista	Pippali, jatiphala, kankola, ela, tvak, tejapatra, nagakesara, lavanga, marica.	[40]
5	Balarista	Payasya, eranda, rasna, ela, prasarani, lavanga, usira.	[14]
6	Dasamularista	Kankola, jala, sveta candana, jatiphala, lavanga, tvak, ela, tejapatra, nagakesara, pippali, kataka phala.	[14]
7	Draksarista	Tvak, ela, tejapatra, nagakesara, priyangu, marica, pippali, vidanga.	[14]
8	Draksasava	Jati, lavanga, kankola, lavali phala, sveta candana, pippali, tvak, ela, tejapatra.	[14]
9	Jirakadyarista	Sunthi, jatiphala, mustaka, tvak, ela, tejapatra, nagakesara, yavani, kankola, lavanga	[14]
10	Kanakasava	Pippali, nagakesara, sunthi, bharngi, talisapatra.	[14]
11	Khadirarista	Kankola, nagakesara, jatiphala, lavanga, tvak, tejapatra, pippali.	[14]
12	Kumaryasava	Jatiphala, lavanga, kankola, jatamamsi, cavya, eranda, karkatasrngi, bibhitaka, puskara.	[14]

 

Apart from basic ingredients the preparation of asavarista formulation require following parameters:

 

Sandhana Patra (Vessels or Containers):

Different terms are used for the containers which have been found in ‘Brihattrayee’, i.e Bhanda, Kalasa, Kumbha, Ghata, Bhajana Patra, Ghrta Bhavita Patra, Madhulipta Patra, Lohapatra, Tamrabhajana, Sucibhajana etc. These containers are made up of earth, wood or metals such as iron and copper. These should be strong enough to withstand long duration of processing⁸. Ancient texts recommend mostly earthenware vessels for asavaristas preparation due to its easy availability, cheap, controlled temperature and inert in nature. However, earthenware vessels have major disadvantages that there is the chance of breakage, oozing of water which leads to loss of yield and makes the formulation more viscous. Nowadays steel and plastic containers are used for fermentation in the pharmaceutical industry¹⁰. Literature reveals that porcelain pot yield more as compared to earthen pot, which was observed in formulation of Arjunarista. Swarna (Saraswatarista) and other metallic containers are also recommended for the preparation of Sandhana kalpana^{41, 42}. Wooden pots are mentioned for fermentation in some classics texts but it has some drawbacks that wooden pots need pre-treatments as it affects the absorption of liquids. There are also some other containers used such as iron for Madhvasva, stone for Kumariasava, etc^{43, 11}. The use of different containers in the preparation of asava and arista are given in (Table 6).

 

 

 

 

Table 6. Sandhana patra used in the preparation of asava and arista

Sl.No	Sandhana patra	Limitations	Example	References
1	Earthen pot	Earthen pots may easily break, oozing of water which leads to loss of yield, makes the formulation more viscous.	Arjunarishta	[42]
			Ashokarista	[44]
			Ashwagandharishta	[45]
			Candanasava	[46]
			Dasamularishta	[47]
			Kutajarista	[48]
2	Wooden containers	It makes the product denser due absorption of liquid by wood. So it requires pre-treatment.	Kutajarista	[48]
			Arjunarishta	[14]
3	Plastic containers	Not mention	Kutajarista	[48]
4	Steel containers	Physico-chemical parameter does not show variation, can be used for large scale production	Kutajarista	[48]
			Ashwagandharishta	[49]
			Dasamularishta	[47]
			Kharjoorasava	[50]
5	Iron patra	Chemical interaction may be occurred between the metallic container and liquid media.	Madhvasva	[43]
6	Swarna patra	Not mention	Saraswatarishta	[41]
7	Porcelain pot	Not mention	Arjunarishta	[42]
			Dasamularishta	[47]
			Kanakasava	[51]
			Kanakbindvarishta	[29]
8	Stone patra	Not mention	Kumariasava	[11]
9	Glass container	The preparation become more acidic than earthen pot	Saraswatarishta	[52]
			Draksharishta	[53]
			Drakshasava	[54]

 

Treatment of Sandhana Patra:

Traditional method reveals that vessels used for fermentation process are to be cleaned by the processes of dhoopana (medicinal fumigation) and lepana (coating or smearing the inner wall of the vessel with medicines). Earthen pots are subjected to Lepan Samskara (coating/smearing) using cow’s ghee to reduce porosity, prevent contamination and the loss of yield and stabilizes the temperature^20,55. Generally ghee, honey or cow’s urine are used as base with herbs like pippali (Piper longom), chavya (Piper retrofractum), priyangu (Callicarpa macrophylla) which made into the form of a paste. Some other drugs like pippali mula, citraka, kamala, agaru, chandana, kustha, vidanga and rala are also used. Lepana is followed by Dhoopana (fumigation), the process prevents the growth of naturally occurring microorganisms which may contaminate or hamper the process of fermentation^35,56. The drugs such as Guggulu, Jatamansi, Vacha, Nimbatwak, Chandan, Agaru, Shweta Sarasapa, Maricha, Karpura, Sarkara, Rala, and Guda are commonly used for fumigation. Amlavirodha (treatment against acid) is carried out for the reused vessel⁵⁷.

 

Sandhana Kriya (Fermentation process):

In the fermentation process measured quantity of dravyas are mixed properly in the dravadravya. Then it is poured into the well-prepared and recommended container having other required ingredients and kept for a specified period of time in suitable place for the purpose of alcohol fermentation. Head space should be left vacant before filling (Purana) of the liquid medium/wort into the fermented vessel. This head space provides space for flashing, foaming and aeration of the liquid. It occupies a fifth to a quarter or more of the volume of the fermenter. Sandhi Bandhana (sealing) of patra is to be done after ensuring onset of fermentation by winding around a long ribbon of cloth smeared with clay on one surface. Sealing is done in such a way that the blank surface of the ribbon will line over the rim of vessel and lid, and the clay side should remain external. Then the vessel is placed in a dark place without much circulation of air. Test is carried out after the completion of fermentation to ensure whether the fermentation is complete or still going on^35,38. After maintaining a specific period of time fermentation is stopped and fermented liquid is tested for the development of necessary organoleptic character in the formulation, which is then filtered and kept for some time to allow the sediments to settle down at the bottom. Supernatant clear liquid is collected and fermented liquid is ready for use⁸.

 

Sandhana Sthala (fermentation place/location):

According to classics, fermentative vessel should be kept in open space or kept in Dhanyarashi (heap of paddy), Dhanya Madhya (Keeping inside the heap of cereals), Yavapalla, and Sugupta sthana (isolated place), ‘Tusa madhya’ etc^58,59. It maintains uniform temperature during Sandhana Kriya (fermentation) because the temperature of Dhanyarashi is more than room temperature and remains constant. Sodhala has showed some new places like Bhugarbha (underground), Suryatrapa (under sun), Koshthasara etc⁶⁰. Yogendra Chintamani added one new place for sandhana i.e Ashwashala (horse shed) for Jambirdrava. Usually the temperature between 25⁰C-30⁰C is considered as ideal for proper fermentation¹⁰. Air conditioner is used nowadays to maintain uniform temperature in most commercial manufacturing set up. As bacteria for fermentation are light sensitive, they are avoided from direct contact of light and air during the storage.

 

Sandhana Avadhi (Duration of fermentation):

The completion of the fermentation reaction depends upon Desha (country or place), Ritu (season) and Dravya (drug substance). Fermentation period varies from 7 days to 6 months due to different nature of constituents, amount of sweetening agents, place, season and use of fermentation initiators etc³⁵. The maintenance of minimum and maximum duration of fermentation as per various classics is mentioned in (Table 7). Some preparations contain iron as the metal source; in this case the maximum time limit advised is till the metal dissolve completely in the solution⁸. Vasa is a major ingredient of Vasarishta which may take short duration for formulation, but in case of Gugguluasava there are many ingredients and guggul being a resin, it may take longer period to get transformed into Asava. So according to nature of ingredients variation in duration of fermentation is observed in different formulations^61-63.

 

Table 7: Duration of Sandhana kalpana

Sl. No	Name of Treatise	Minimum period of duration	Example	References	Maximum period of duration	Example	References
1	Caraka Samhita	7 Days	Vasarishta	[64]	1/2 Month	Arjunarista	[25]
2	Susruta Samhita	7 Days
3	Astanga Sangraha	7 Days			6 Months	Guggulasava	[65]
4	Astanga Hrdaya	15 Days	Arjunarista	[25]	1 Month	Ashwagandharista	[66]
						Kanakasava	[51]
						Saraswataristam	[67]
						Kutajarista	[48]
						Saraswatarishta	[52]

 

Finished product:

In Sanskrit it is called as ‘Jaatarasam’ which denotes completion of fermentation process to form asava and arista⁶⁸.

 

Filtration of asava-arista:

The fermented product is filtered through double layered clean cotton cloth to separate the sediments after completion of fermentation process. The filtrate is allowed to stand for few more days and again filtered to ensure the separation of sediments, if required.

 

Nirmalinikaran (Cleansing of Asava-Arista):

Nirmalikaran is carried out by spreading coarse powder of Kataka (Strychnos potatorum) on top of the filtrate which absorbs the remaining sediments and produce clean final liquid product⁶⁹.

 

Storage:

After their preparation the fermented product asavas and aristas are stored in dark coloured vessels to avoid direct exposure to light.

 

CONCLUSION:

Asava and arista formulations are considered as unique preparation in ayurvedic medicine due to their longer shelf life period, which could be due to generation of alcohol by fermentation. In the present review different parameters are discussed, which are essential for the preparation of these unique formulations. Several traditional earthenware pots were used for the preparation of asavarista which are being replaced by plastic and steel tank in modern technology. Some pharmacies use yeast as fermentative agent in place of traditional Dhataki puspa. There are lot of variations observed with respect to percentage of madhura dravya and prakshepa dravya used in different types of asavarista formulations. Duration of fermentation process varies according to place, season and nature of ingredients. In modern technology air conditioned room is used as sandhana sthala for large scale production. Nowadays instead of using traditional methods, non-traditional methods are being followed for formulation of asava and arista to minimise the duration of preparation. Using the same drug of traditional asavarista formulation, the syrupy preparations are being produced comparatively within a short time period. However, by adopting the non-traditional methods of preparation, it would be a matter of great concern of maintaining the quality and efficacy of these ayurvedic formulations.

 

ACKNOWLEDGEMENT:

The authors are grateful to the Dean of School of Pharmaceutical Sciences, Siksha ‘o’ Anusandhan Deemed to be University for providing all facilities and encouragement throughout the work.

 

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 02.11.2018         Modified on 15.01.2019

Accepted on 28.01.2019      © RJPT All right reserved