Comparative Assessment of Phyto and Physico-chemical Parameters of Laboratory prepared Two Renowned Samples of Polyherbal Formulation “Arq Aswad Barid” by Adopting Two Different Methods (Classical and Modern) for the Step of Fermentation Process with the Preliminary Study of Its Ingredients

 

Akhlaq Mustafa¹*, Mushtehasan², Kiran Negi³, Anas Iqbal Alvi⁴, Ghazala Javed⁵

¹Research Officer (Chemistry), Drug Standardization Research Unit,

Central Council for Research in Unani Medicine, New Delhi.

²Research Officer (Unani), Drug Standardization Research Unit,

Central Council for Research in Unani Medicine, New Delhi.

³Assistant Research Officer (Pharmacognosy) Drug Standardization Research Unit,

Central Council for Research in Unani Medicine, New Delhi.

⁴Research Assistant (Chemistry), Drug Standardization Research Unit,

Central Council for Research in Unani Medicine, New Delhi.

⁵Research Officer (Unani), Incharge, Drug Standardization Research Unit,

Central Council for Research in Unani Medicine, New Delhi.

 

ABSTRACT:

There is an increasing awareness and general acceptability of indigenous drugs in today’s medical practice and over 80% of the world population depends on herbal drugs and products for healthy living due to their no side effects. Unani formulations are based on herbal, mineral, and animal origin and have been practiced in India for hundreds of years for treating various ailments by its holistic approach with natural means and drugs. Research is a creative work using existing knowledge for further development and advancement of knowledge and the approach, methodology and technique of research should be consistent with the ultimate objective because the scientific method of research is a combination of hypothesis, experimentation, observation, and reasoning. Therefore, the criteria of real scientific research are that its result should be reproducible under similar conditions. The paper deals with the objective of standardization and makes experimentation, and observation and particularly to check out Physico-chemical data of two in-house prepared samples of compound Unani formulation ArqAswadBaridunderSOP guidelines by adopting two different methods(classical and modern) of fermentation process separately and the content so obtained after distillation from fermented materials were utilized further to prepare two separate samples of the finished products i.e. Arq-e-Aswad Barid and underwent for the comparative assessment by following some Physico-chemical and quality control parameters like viscosity, specific gravity, refractive index, weight per ml, detection of aflatoxin, microbial load, pesticide residue, etc. of both the samples to achieve the objective of study. Besides, the preliminary physicochemical study of all ingredients used as raw material in the preparation of Arq-e-Aswad Barid was also conducted.

 

 

INTRODUCTION:

Unani medicines have long been used in India and abroad for various disorders and diseases. Arqiyat is a traditional Unani liquid preparation, prepared by simple distillation for single and combination of medicinal plants which have a major source of treatment and are a promising choice over modern synthetic drugs. They show minimum or no side effects and are considered to be safe. It is a form of the drug in which the distillate is obtained in the form of vapors, which are collected after condensation. It is a volatile part of drugs in the form of liquid. It is an important and exclusive dosage form of the Unani system of medicine. Arq preparations are mentioned in the Unani pharmacopeia of India and have been used for various therapeutic purposes such as liver disorders vomiting abdomen pain palpitation and inflammation^1,2, etc.

 

The general method for herbal drug preparation includes three phases³. The extraction process is the most important step in the preparation of the herbal drug. During this step, bioactive and nutritive molecules present in the herbs are released into the medium. There are many other modern extraction techniques available like mashing, maceration, solid-phase extraction, fermentation, etc. Among these techniques, fermentation (Takhmir) is gaining interest in the scientific community to produce novel active compounds with potent medicinal values during herbal drug preparation^4. As far as fermentation is concerned, it is a process of chemical change caused by microorganisms for transformation into another compound of medicinal value. During fermentation, the plant cells rupture more efficiently, and hence a wider range of active ingredients are exposed to the menstruum. The alcohol produced during the fermentation process helps in releasing the compounds from the cells to the medium^5,6. The microorganism used in the fermentation releases various types of enzymes that disintegrate the herbal medicines or the target molecules and convert them into new entities of medicinal value.⁷ In addition, microorganism uses active herbal ingredients as substrates or secondary metabolites produced during fermentation to interact with each other to produce new compounds or modify the naturally occurring molecules such as isoflavones, saponins, phytosterols, and phenols and thus exert more therapeutic potencies and lowers the toxicity. The non-medicinal components, such as proteins, sugars, and other substances are also removed during fermentation making the herbal drugs more bio-available and safe^8,9.

 

In the Unani system of medicine, various products have been mentioned in classical literature where the process of fermentation is applied for the preparation of drugs like Sirka, Nabeez, Dar Bahra, Aabkaama, etc. The preparation of sirka is well described in the Unani system of medicine. A microorganism called Ummulkhal (mother of vinegar) or mycodermaaceti plays a key role in the process of fermentation¹⁰. However, in ancient times, the involvement of a microorganism in the process of fermentation may be unknown. Sirkapossesses anti-infective, anti-hypertensive, cardio-protectant-tumor, and anti-oxidant activities¹¹. Aabkaama, Muri, and Kanji are also liquid preparations made up of Rae, Namak, and Ajwain with the same procedure as Sirka. Sikanjabīn is also prescribed in Unani literature made with vinegar and honey but later honey was substituted with sugar. It is of many types like SikanjabīnAsuli (roots), SikanjabīnBazuri (seeds), Sikanjabīnlemuni (Lemon), Sikanjabīnunsuli (Piyaz), and Sikanjabīnfawakiha (Fruits), etc^12-16.

 

Nabeez is a dosage form used in the earlier era of Unani medicine obtained by the fermentation of sweet aqueous solutions of various food grains, fruits, and medicinal ingredients for a specific period. There are many types of Nabeez formulations mentioned in Unani literature mostly named against their chief constituent. It is prepared by keeping the decoction of the herbal drug in the form of a solution of sugar or jaggery in a container whereas, in Dar Bahra, the cold infusion of the drugs is used to make the solution with Sugar, Jaggery, and Honey, etc. The fear of intoxication in manufacturing methodology makes Nabeez and other fermented Unani formulations unpopular in India. The previous study showed that the modification in the manufacturing procedures of Nabeez is beneficial for the improvement of the quality of the product and the economy¹⁷.

 

MATERIAL AND METHODS:

The formulation “Arq-e-Aswad Barid” consists of 20 ingredients and all belong to the plant origin. These ingredients were procured from a local raw drug dealer and identified botanically^18,19 using pharmacognostic methods. The ingredients were further validated by comparing them with the monograph available in Unani Pharmacopoeia of India (UPI) and Ayurveda Pharmacopeia of India (API)²⁰. All the ingredients taken were of pharmacopoeial quality. The ingredients were cleaned and dried under shade to remove moisture. The drug was prepared as per the formula mentioned in NFUM, Part-1 2006 Ministry of Health and Family Welfare, Govt. of India²¹.

 

 

Table No. 1 Formulation Composition

 

Method of Preparation:

For the preparation of the drug, the first step is to prepare the fermentation material by mixing the ingredient serial no.1 and 2 in an earthen or wooden vessel filled with 2/3 rd of distilled water and sealed it. Now the fermentation process was completed by following two methods separately.

 

First method (Classical):

The above whole sealed vessel containing ingredient serial no.1 and 2 was now placed in a large pit in the ground and covered all around with cow dung cakes till the fermentation was complete. It was taken out from the pit of the ground after 12 days. The Arq (Distillate) is then collected as usual by transferring the whole content of fermented material into a distillation flask.

 

Second method (Modern):

In this method, the whole vessel containing ingredient serial no.1 and 2 was filled with 2/3^rd of water along with the addition of a sufficient quantity of yeast (Saccharomyces cerevisiae) for fermentation and sealed properly and was now placed into a thermostatically controlled oven at temperature 35⁰C for approximately seven days or till complete the fermentation process. The Arq (Distillate) is then collected as usual by transferring the whole content of fermented material into a distillation flask. After completing this step of the fermentation process for both samples separately the same method was followed to get the finished product described as under.

 

Now crush ingredients no. 6, 7, and 14. After crushing, mix ingredients no 3-19. Soak all the ingredients in Arq of ingredients no.1 and 2 and leave them overnight. Transfer the soaked material into the distillation unit. Distill the soaked material at near boiling temperature. Collect the Arq in glass containers as a finished product of Arq-e-Aswad Barid and seal them tight. In this way, two samples of AAB were prepared by following two different methods of fermentation named classical and modern methods AAB (C) and AAB (M) respectively.

 

Analysis of Physico-chemical parameter:

pH values:

This is a very important parameter regarding the point of standardization of the drug which tells us whether the drug has an acidic, alkaline, or neutral character. pH as such of both the finished product samples as well as the distillates of fermented products (prepared by classical and modern methods) was measured with pH meter^22-23.

 

Refractive Index:

The refractive index of a substance with reference to air is the ratio of the sine of the angle of incidence to the sine of the angle of refraction of a beam of light passing from air into the substance. It varies with the wavelength of the light used in its measurement²⁴. Determination of the Refractive index was measured by using Abbe’s Refractometer.

 

Viscosity:

Viscosity is a property of a liquid, which is closely related to the resistance to flow. The viscosity of samples was determined by the U-tube viscometer^25-26. (Anonymous, 1986).

 

Specific gravity:

The specific gravity of a liquid is the weight of a given volume of the liquid at the specified temperature compared with the weight of an equal volume of water at the same temperature, all weighing being taken in air. For the determination of specific gravity, a 25ml pycnometer was taken. It was weighed accurately, previously cleaned and dried, and the weight ‘W’ was noted. The stopper was removed, and the pycnometer was filled with the market sample at 25ºC temperature^27-29(Ueda et al, 2009).

 

Table No. 2: Physical/Chemical tests

 

Weight per ml:

It was determined by dividing the weight in the air, expressed in the gram of the quantity of liquid that fills a pycnometer at a specified temperature by the capacity, expressed in ml of the pycnometer at the stated temperature²⁶.

 

Physical observations:

Appearance:

The appearance of the drug tells us about the state of its consistency whether it is liquid, semi-liquid, semisolid, solid, etc.

Color: Samples were observed physically with the naked eye to know their color.

Odor: Theodor was examined by repeated and slow inhalation of air over the samples by the different healthy people having a good sense of smell.

Taste: It was determined by asking different healthy people to taste the samples one by one.

 

Observations and Results:

Fermented materials analysis:

Before adding the rest of the ingredients of AAB in the distillate of fermented material some physical/Chemical tests were conducted as given in the following table no 2

 

Chemical analysis of finished products AAB(C) and AAB(M)

(a). Organoleptic characters:

Organoleptic evaluation of both the samples of Arq-e-Aswad Barid is given below as in table no.3.

 

(b). Physicochemical analysis:

Both samples were analyzed for some physicochemical standards. The values so obtained are given in Table No. 4.

Table No. 3: Organoleptic Characteristics

 

 

Table No. 4: Physicochemical Standards

 

(c) Qualitative analysis of phytoconstituents^30-34:

In the qualitative phytoconstituent analysis, different chemical tests of various constituents were carried out. Test of terpenoids, flavonoids, and alkaloids was positive while the presence of essential oil is in traces in both cases, and carbohydrates, saponins, glycosides, tannins and proteins were found to be negative as given in the following table no. 5

 

(d). Quality control parameters:

In the quality control analysis some parameters like microbial load, aflatoxins, and pesticidal residues for both the samples of finished products were conducted by following the guidelines and as per the prescribed methods in the books ^35-37 (WHO, 2007), AOAC (2000and2005) respectively, and presented in table nos. 6, 7, and 8 as given below.

 

 

Table No. 5: Phytochemical evaluation

 

Table No. 6: Analysis of Microbial Load

 

Table No. 7: Estimations of Aflatoxin

 

Table No.8: Estimations of Pesticide residue

*BLQ-Below Limit of Quantification/ LOQ- Limit of Quantification

 

Physico/ phytochemical analysis of the ingredients used in the preparation of Arq-e-AswadBarid:

A preliminary study of physico-chemical standards was conducted of all the ingredients used in the preparations of Arq Aswad Barid and The results obtained are given in Table No. 9.

 

Table No. 9: Physicochemical standards of components of AAB

 

DISCUSSION:

Fermentation is an ancient process that has been used to improve taste, texture, and color, and relies on pH, among many other factors like the absence or presence of air, temperature, and water which is one of the most important chemical environmental measurements used to indicate the course of the fermentation process. It detects the presence of specific chemical factors that influence growth, metabolism, and final product. For yeast, fermentation will begin at 75° to 85°F (23.9° to 29.4°C). The yeast cells used in fermentation can tolerate a pH of 4.0 to 8.5 but work best when the pH is between 4.0 and 6.0. This means that yeast cells require a slightly acidic environment to do their best fermenting. The pH values of the distillates of both fermented products using classical and modern methods were found to be 4.35 and 4.21 (Table no. 2) respectively.

 

The drug composition contains 19 components. All are of plant origin and have great medicinal values due to the presence of many important secondary metabolites like alkaloids flavonoids, and triterpenes as can be seen in the analysis given in Table no. 9. Arq Aswad Barid has Mufarreh (exhilarant), and Musakkin (soothing) actions and is useful in Malikhuliya (melancholia), Miraq (hypochondriasis), Khafqan (palpitation). The exhilarant and soothing actions may be due to the presence of Sandal Safaid (Santalum album L.), Gul-e-Nilofar (Nymphaea alba L.), Gul-e-Gaozaban (Boragoofficinalis L.), and Bed Sada (Salix alba L.)^{38- 40}. These plant materials contain various important Phyto-constituents like Alkaloids, flavonoids, and phenolic compounds. Therapeutically, alkaloids include morphine, strychnine, quinine, ephedrine, and nicotine^41. are particularly well-known as anesthetics, cardioprotective, and anti-inflammatory agents. Flavonoids possess several medicinal benefits, including anticancer, antioxidant, anti-inflammatory, and antiviral properties. They also have neuro-protective and cardio-protective effects. Natural phenolic compounds play an important role in cancer prevention and treatment. Various bioactivities of phenolic compounds are responsible for their chemopreventive properties (e.g., antioxidant, anti-carcinogenic, or anti-mutagenic and anti-inflammatory effects)^42,43,

 

Evaluation of organoleptic characteristics refers to the drug’s appearance, color, odor, taste, etc. Observations of these characteristics and the results of physicochemical parameters e.g. specific gravity viscosity refractive index optical rotation etc. of the fermented material distillates obtained from classical and modern methods are given in Table no. 2 where the data in both cases are nearly similar. The absence of sugar and the presence of alcohol in the test solutions of both fermented products indicate that complete and proper fermentation took place during the process. Again, all the organoleptic characteristics and the physicochemical findings of both the samples of the finished products, AAB(C) and AAB(M) prepared by two separate methods can be seen in Tables No. 3 and 4. The pH values of AAB(C) and AAB(M) were found to be 3.43 and 3.10 respectively. So the data obtained in all the parameters did not show any considerable variations and are almost the same in both samples. The quantitative analysis of the quality control parameters is mentioned in Table nos. 6, 7 and 8, which indicate that the microbial load was within the WHO permissible limit, aflatoxins content (B1, B2, G1, and G2) was absent, and also, the drug was found to be free from pesticide residues contamination in both cases. So it can be stated that the drug is quite safe for internal use.

 

CONCLUSION:

The main objective of the study was to check out the difference in the morphological characters and the Physico-chemical data of the distillates of fermented products and two in-house prepared samples of compound Unani formulation ArqAswadBaridin which two different methods of fermentation process were adopted. In fermentation (TAKHMEER) sugar is converted into alcohol and in case the fermentation process is incomplete or not proper, then it would certainly reflect on the results of the parameters by showing some sort of deviations. The above-mentioned results in physical parameters and Physico-chemical analysis data of fermented products as well as the finished products did not show any significant variations in both the samples prepared by adopting different methods of fermentation and findings were similar and found to be satisfactory. Therefore, as per our study, it can be concluded here that any method described above can be utilized to complete the step of the fermentation process. However, the modern method is a little bit hassle-free and has no need to wait for any conducive atmosphere or changes in weather conditions and can be implemented by using electric equipment for maintaining the time and temperature. So it looks convenient. Besides, these findings may also be used as the standards for ensuring the purity, quality, and authenticity of the drug.

 

ACKNOWLEDGMENT:

The author is thankful to the Director General CCRUM for providing support and necessary facilities for the study.

 

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Received on 03.09.2023         Modified on 13.09.2023

Accepted on 22.09.2023   ©Asian Pharma Press All Right Reserved