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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article                                      Research Article

Standardization and Development of Standard Operating Procedures (SOPs) of a Polyherbal Unani Formulation Qurṣ-i Bawāsīr

¹ Azizur Rahman, *² Shagufta Nikhat, ³ Javed Inam Siddiqui, ⁴ Uzma Viquar, ⁵ Mohd. Abdul Rasheed Naikodi, ⁶ Younis Iftikhar Munshi

¹Postgraduate Research Scholar, Department of Ilmul Advia, National Research Institute of Unani Medicine for Skin Disorders, Hyderabad

²Professor and HOD, Department of Ilmul Advia, National Research Institute of Unani Medicine for Skin Disorders, Hyderabad

³Professor, Department of Ilmul Advia, National Research Institute of Unani Medicine for Skin Disorders, Hyderabad

⁴Professor, Department of Ilmul Advia, National Research Institute of Unani Medicine for Skin Disorders, Hyderabad

⁵RO Chemistry, DSRU, National Research Institute of Unani Medicine for Skin Disorders, Hyderabad

⁶Incharge Director, National Research Institute of Unani Medicine for Skin Disorders, Hyderabad

 

 

INTRODUCTION

The term Unani is derived from the Greek word Ionian which means the awareness of the health and disease states of the human body. The Unani system of Medicine emerged principally from Greece and brought by Arab and Persian pioneers in India and it embraced worldwide, due to its easy accessibility, inexpensiveness and comparatively safer than other traditional medicines. The World Health Organization (WHO) is the predominant and specialized agency that had a direct influence on global healthcare and acknowledged Unani System of Medicine in 1976 ^1. The health and disease discourse initiated around 500 BC with the influential Greek philosopher and physician Hippocrates, Father of Medicine liberated the medicine from magical and supernatural beliefs, advocating for a rational approach. He emphasized on natural causes of diseases and conducted experiments to demonstrate the body's natural reactions to illness. The Hippocratic Corpus, devoid of mystical references, showcases his data-driven approach ^{2, 3}.

The most common reasons for using traditional medicine are that it is more affordable; more closely corresponds to the patient’s ideology, allays concerns about the adverse effects of synthetic modern medicines, satisfies a desire for more personalized health care, and allows greater public access to health information ⁴. In the present era, universal trend has been shifted from synthetic to herbal medicine i.e. Return to Nature ⁵. Unani is a time-tested, trusted, plant-based system of medicines which is developed through everyday life experiences with the mutual relationship between mankind and nature ^{6, 7}. As per WHO, there are three -kinds of herbal medicines: raw plant material, processed plant material and medicinal herbal products ⁸. 

It is no wonder that the world’s one-fourth population is using traditional medicines for the treatment of various ailments ⁹. However, one of the impediments in the acceptance of the Unani medicines is the lack of standard quality control profiles ¹⁰. Due to the complex nature and inherent variability of the chemical constituents of the plant-based drugs, it is difficult to establish quality control parameter ¹¹. 

Quality assurance of herbal medicine is an important factor and basic requirement for herbal drug industry and other drug development organization ¹². Standardization is a system that ensures a predefined amount of quantity, quality and therapeutic effect of ingredients in each dose. Herbal product cannot be considered scientifically valid if the drug tested has not been authenticated and characterized in order to ensure reproducibility in the manufacturing of the product. Moreover, many dangerous and lethal side effects have recently been reported, including direct toxic effects, allergic reactions, effects from contaminants, and interactions with herbal drugs.

Therapeutic activity of a herbal formulation depends on its phytochemical constituents. The development of authentic analytical methods which can reliably profile the phytochemical composition, including quantitative analyses of marker/ bioactive compounds and other major constituents, is a major challenge to scientists. Standardization is an important step for the establishment of a consistent biological activity, a consistent chemical profile, or simply a quality assurance program for production and manufacturing of an herbal drug. The authentication of herbal drugs and identification of adulterants from genuine medicinal herbs are essential for both pharmaceutical companies as well as public health and to ensure reproducible quality of herbal medicine ⁸.

The study drug Qurṣ-i Bawāsīr is one of the most crucial Unani formulations which has been mentioned in several Unani classical books. This study formulation is taken from Qarabadeen-i Majeedi ¹³. It has synergistic effects in the treatment of Bawāsīr (Piles). Its ingredients act as anti- inflammatory, analgesics, purgative and emollient etc. There is no sufficient data on drug standardization and quality control of Qurṣ-i Bawāsīr, therefore, to establish the quality, standardization, authenticity, and using modern analytical techniques, the study drug Qurṣ-i Bawāsīr is identified and selected for this study. Considering the above circumstances, an attempt to complete documentation on the compound formulation has been made in which we performed standardization of Qurṣ-i Bawāsīr, which could be beneficial in the future for preparing the monograph.

MATERIALS AND METHODS

Collection of material: The ingredients of the Polyherbal formulation "Qurṣ-i Bawāsῑr" were procured from the GMP certified pharmacy, identified and authenticated by the botanist at National Research Institute of Unani Medicine for Skin Disorders, Hyderabad, India. 

Preparation of the Formulation: The study formulation of Qurṣ-i Bawāsῑr has been selected from Qarabadeen-e-Majeedi and prepared in GMP certified pharmacy of NRIUMSD, Hyderabad as per the standard procedure mentioned in the Qarabadeen-e-Majeedi and other reference books and the following SOPs developed in accordance with the preparation of formulation. 

Authentication of ingredients of study formulation: The ingredients of Qurṣ-i Bawāsῑr were identified and authenticated by the botanist in Survey of Medicinal Plants Unit (SMPU), NRIUMSD, Hyderabad and a voucher specimen number for Neem- SMPU/CRI-Hyd14999, Guggul- SMPU/CRI Hyd15000, Rasaut- SMPU/CRI- Hyd15001, Reetha SMPU/CRI-Hyd 15002, Samagh-i Arabi SMPU/CRI-Hyd 15003 were allotted and deposited in herbarium.

 

 

S. No.	Ingredients	Botanical Name	Part Used	Quantity
1.	Muquil	Commiphora mukul	Dried Resin	125 g
2.	Rasaut	Berberis aristata DC.	Root extract	125 g
3.	Neem	Azadirachta indica A. Juss.	Seed’s Kernel	30 g
4.	Reetha	Sapindus laurifolius Vahl.	Dried pericarp of fruit	30 g
5.	Samagh-i-Arabi	Acacia nilotica (L)	Gum	60 g

 

 

According to pharmaceutical procedure and standardization, the conventional and modern analytical techniques were used to standardize "Qurṣ-i Bawāsῑr" (QB). In this study three batches of QB were prepared by standard method as per Qarabadeen-e-Majeedi had been followed by organoleptic properties of formulation such as appearance, color, odor, taste. Powder Microscopy and physicochemical studies were carried out such as uniformity of weight, friability, disintegration time, hardness, LOD, pH of 1% and 5% aqueous solution, ash values and extractive values in different solvents like aqueous, alcohol & hexane. Further qualitative tests such as Thin Layer Chromatography (TLC), and High Performance Thin Layer Chromatography (HPTLC) studies were also carried out to develop fingerprint pattern of the alcoholic solvent extract of QB. Phytochemical screening was carried out in different solvent extracts such as alcoholic, aqueous and chloroform extracts to detect the presence of phytoconstituents in the formulation QB. Heavy metals, microbial load contamination, aflatoxins and pesticidal residues were also determined. QB was analyzed for heavy metal on an Atomic Absorption Spectrophotometer at DSRI, Ghaziabad. Flame atomization has been applied for detection of Lead (Pb) & Cadmium (Cd) and Hydride generator was used for the detection of the elements Arsenic (As) & Mercury (Hg) ¹⁴. The microbial load analysis was carried out in three different samples of QB. The media used are Soyabean Casein Digest Agar Media, Sabouraud Dextrose Agar with Chloramphenicol Media, HiCrome TM E. Coli Agar Media, Hi Crome Raj Hans Medium, modified (Salmonella Agar, Modified). Aflatoxins can be harmful to health even though they are consumed in minute quantities in herbal medicines. This research was performed to identify the possible presence of B1, B2, G1 and G2 aflatoxins in QB¹⁴. QB was analyzed for pesticidal residue at Bureau Veritas Testing laboratory, Hyderabad. The procedure and methods for analysis were followed on the protocol of WHO issued guidelines¹⁴.

Thin Layer Chromatography:

Thin layer chromatography was used for qualitative screening of QB extract for separation and determining number of compounds present in the solvent extract. Thin layer chromatography was carried out on pre coated Aluminium plates, silica gel 60 F254 (layer thickness 0.25 mm). The extracts of QB were applied to the plates (1 cm above from the bottom). It was then kept in previously saturated developing chamber containing mobile phase, and allowed to run up to 80 mm of the height of the plate. The developed plate was then removed, air dried and observed under ultraviolet light and florescent compound was observed and calculated the Rf value for each spots using following formula.

R_f Value = Distance travelled by spot/Distance travelled by solvent

High Performance Thin Layer Chromatography (HPTLC) Analysis:

HPTLC is one of the important and widely used methods for separation as well as quantitative estimation of marker compounds present in herbal drugs. HPTLC finger print profile is the best choice for standardization     of components followed by determination of phyto-constituents from drug extracts. The HPTLC fingerprint for the formulation was developed. Major advantage of HPTLC is its ability to analyze several samples with minimal quantity of sample.

5 grams of powdered QB is taken and reflux with 200 ml of ethyl acetate using Soxhlet apparatus    on a water bath for 30 min. Filter the extract and concentrate to 5 ml then the sample extract obtained was used for high performance thin layer chromatography

RESULT AND DISCUSSION

In this research work, standardization of Qurṣ-i Bawāsῑr (QB) was carried out in terms of its physicochemical, phytochemical and safety profile. The physicochemical study of QB includes the parameters such as organoleptic properties of formulation such as appearance, color, odor, taste and physicochemical studies were carried out such as uniformity of weight, friability, disintegration time, hardness, LOD, pH of 1% and 5% aqueous suspension, ash vales and extractive values in different solvents like aqueous, alcohol & hexane. Further tests such as Thin-Layer Chromatography (TLC), and High-Performance Thin Layer Chromatography (HPTLC) studies were also carried out to develop fingerprint pattern of the alcoholic solvent extract of QB. Phytochemical screening was carried out in different solvent extracts such as alcoholic, aqueous and chloroform extracts to detect the presence of phytoconstituents in the formulation QB. Heavy metals, microbial load contamination and pesticidal residues were also determined.

Quality assurance is an important part of all systems of medicine to establish the standard quality pharmaceutical drugs. Thus, there is high-priority requirement for the evaluation of parameters that can be adopted by the pharmaceutical industries for quality assessment of traditional preparations. Powder microscopy was done for the detection of microscopic structures present in that specific drug. The efficacy of a drug mainly depends upon its physical and chemical properties, so need of physicochemical characters is necessary for authenticity of the drug. Physicochemical study is also important, because it helps in characterization of constituents or groups of constituents that frequently lead to establish the structure activity relationship as well as mechanism of drug action. The phytochemical constituents present in the drug may vary, not only from plant to plant but also among the different sample of same species, depending upon various atmospheric conditions, storage, and drying conditions. If there is any deviation in terms of quality and quantity, that may alter the efficacy of the drug. In spite of quality assurance, adulteration is another factor that may contribute to variability.  

Organoleptic evaluations: Qurṣ-i Bawāsīr was found blackish brown in color due to presence of Muqil and Rasaut. Shape of the tablet was flattened disc shaped. Odor was characteristic, due to presence of Maghz-i Neem (Neem seeds) and Reetha. Taste was found to be bitter and texture was slightly rough. Organoleptic evaluation is an essential parameter for fast identification and consumer acceptance. Poor organoleptic properties not only lack aesthetic appeal and non-uniformity of content.  

Microscopic study of Powder: The microscopic features of QB were observed and the powdered drug was found to be light blackish brown in color. An abundance thin-walled outer epidermal cells were present in surface view, due to the presence of fibers and stone cells. Fibers were long, lignified with small lumen and stone cells were lignified, mostly rectangular in shape. It showed fragments of tissues; cork cells were present. Cork cells are slightly thickened wall containing reddish brown contents. Cells containing groups of calcium oxalate crystals and sclereids are observed in powder microscopy (fig. 4).

Uniformity of weight of QB: The mean value of randomly selected twenty tablets of 3 batches for uniformity of weight was found in the range 543±9.11 to 547±8.75 mg. The deviation of individual tablet weight from the mean weight twenty tablets of 3 batches were found within the percentage limit of 5%. The data of uniformity of weight of QB is represented in the table-1

Friability test: The mean percentage of friability values of QB were found in the range of 0.0549 to 0.0673% in three different batches. The data for friability is depicted in the table-1. 

Disintegration time of tablets: The mean value of disintegration time of QB in aqueous media was found 35 minutes in three different batches. The data of disintegration time is depicted in the table-1.

Hardness test: The mean percentage of hardness of QB was found in the range of 8 to 9 kg/cm² in three different batches. The data for hardness test is depicted in the table-1.

Loss of weight on drying at 105 °C: The mean percentage of loss of weight on drying at 105°C of QB was found to be in the range of 4.2-4.4 % w/w in three different batches. The result for loss of weight is represented in the table-1.

pH of 1% aqueous suspension: The mean percentage pH of 1% aqueous suspension of QB was found in the range of 5.95–5.96 % w/w in three different batches. The obtained data for pH is depicted in the table-1.

Total Ash values: The mean percentage value of total ash of the QB was found in the range of 12.0024±0.358 to 12.3569 ±0.058% w/w in three different batches. The resultant data of total ash value is depicted in the table- 1.

The mean percentage of acid insoluble ash value of QB was observed in the range of 5.0511±0.124 to 5.3262 ±0.490% w/w in three different batches. The obtained data of acid insoluble ash is depicted in the table-1.

In present study, a systemic phytochemical screening was carried out in different solvents (aqueous, methanol and chloroform) extracts of the QB and the qualitative test was done and reported the nature of phytoconstituents present in the formulation (table 2).

 

 

Table 1: Physicochemical parameters of the compound formulation QB

Parameters	Batch 1 (Mean ± SD)	Batch 2 (Mean ± SD)	Batch 3(Mean ± SD)
Uniformity weight of tablet (mg)	546±9.45	547±8.75	543±9.11
Friability test (%)	0.0549±0.001	0.0673±0.049	0.0663±0.001
Disintegration Time in aq. medium (min)	35±0	35±1	35±1
Hardness test (kg/cm)	9.1±0.173	8.1±0.173	8.06±0.115
Loss in weight on drying at 105˚C (% w/w)	4.4±0.346	4.2±0.0	4.23±0.152
pH of 1% (aqueous suspension)	5.96±0.034	5.95±0.020	5.96 ±0.015
pH of 5% (aqueous suspension)	5.28 ±0.037	5.32 ±0.041	5.33 ±0.015
Alcohol soluble matter (%w/w)	21.2567 ±1.048	20.4893 ±0.692	20.9009 ±0.930
Water soluble matter (%w/w)	67.2113±0.485	64.9811±1.184	66.7053±0.343
Hexane soluble matter (%w/w)	8.2438±0.068	10.0953±0.362	10.0578±0.155
Total Ash values (% w/w)	12.0024±0.358	12.3569 ±0.058	12.3350 ±0.123
Acid Insoluble Ash (% w/w)	5.0511±0.124	5.1323±0.086	5.3262 ±0.490

 

Table 2: Analysis of phytochemical constituents of QB

Phytochemical constituents	Test	Observation
		Alcoholic extract	Aqueous extract	Chloroform extract
Alkaloid	Dragendroff’s test	+	–	–
	Mayer’s test	+	+	–
	Wagnerʼs test	+	–	+
Carbohydrate	Fehling’s test	+	–	+
Phenols	Ferric chloride test	+	–	+
	Lead acetate test	+	+	–
Proteins	Millon’s test	-	-	-
Flavonoids	Shinoda test	-	-	-
Tannin	Ferric chloride test	+	–	+
	Lead acetate test	+	+	–
Saponin	Foam test	–	+	–
Steroids	Salkowski’s test	+	+	+
Starch	Millon’s test	-	-	+
Glycoside		+	–	–

Table 3: Safety parameters of the formulation QB

 

 

Table 4: Heavy Metals

S. No.	Parameters analyzed	Results	WHO Permissible Limits
1.	Lead-(Pb)	ND	10 ppm
2.	Cadmium-(Cd)	ND	0.3 ppm
3.	Arsenic-(As)	ND	3.0 ppm
4.	Mercury-(Hg)	ND	1.0 ppm

 

HPTLC Analysis of Ethyl Acetate Extract of Study Formulation QB: The ethyl acetate extract of QB was applied on precoated silica gel “G” plate and developed with solvent system Toluene: Ethyl acetate: Methanol (7:2:1 v/v/v) as the mobile phase. The chromatogram showed eight spots under UV 366 nm at Rf values 0.24, 0.35, 0.39, 0.43, 0.52, 0.64, 0.73, 0.97. TLC plate and showed ten spots under UV 254 nm at Rf values 0.27, 0.32, 0.36, 0.41, 0.50, 0.57, 0.65, 0.71, 0.78, 0.95 as depicted in table 5and densitograms (fig. 2, 3).

 

Table-5

Peak list and Rf values for Ethyl acetate extract at 366nm wavelength
Peak No.	Max Rf	Height	Height %	Start Rf	Start height	End Rf	End Height	Area	Area %
1	0.247	0.0669	7.59	0.214	0.0090	0.0263	0.0373	0.00184	6.03
2	0.357	0.0504	5.72	0.341	0.0358	0.373	0.0338	0.00134	4.41
3	0.394	0.0723	8.20	0.373	0.0338	0.414	0.0398	0.00225	7.36
4	0.430	0.0514	5.83	0.414	0.0398	0.469	0.0029	0.00161	5.27
5	0.523	0.1252	14.20	0.510	0.1080	0.574	0.0028	0.00331	10.48
6	0.646	0.0755	8.56	0.603	0.0043	0.701	0.0073	0.00087	9.42
7	0.731	0.0176	1.99	0.701	0.0073	0.780	0.0000	0.00070	2.30
8	0.970	0.4224	47.91	0.929	0.0000	1.004	0.0014	0.01660	54.39
Peak list and Rf values for Ethyl acetate extract at 254nm wavelength
Peak no	Max Rf	Height	Height %	Start  Rf	Start height	End Rf	End height	Area	Area %
1	0.273	0.0852	5.17	0.200	0.0332	0.290	0.0691	0.00529	6.87
2	0.327	0.1181	7.16	0.290	0.0691	0.350	0.0637	0.00524	6.81
3	0.363	0.0754	4.57	0.350	0.0637	0.381	0.0541	0.00210	2.72
4	0.416	0.1545	9.36	0.381	0.0541	0.440	0.0568	0.00566	7.35
5	0.509	0.1476	8.95	0.467	0.0703	0.530	0.0694	0.00711	9.25
6	0.579	0.2364	14.33	0.530	0.0694	0.624	0.0211	0.01400	18.21
7	0.656	0.0850	5.15	0.624	0.0211	0.683	0.0350	0.00329	4.27
8	0.719	0.1498	9.08	0.684	0.0349	0.741	0.0934	0.00600	7.81
9	0.781	0.4520	27.39	0.743	0.0930	0.837	0.0393	0.02043	26.56
10	0.954	0.1461	8.85	0.914	0.1002	1.000	0.0000	0.00780	10.14

   

At UV 366nm                                    At UV 254nm                              After derivatization

Figure 1: HPTLC plate of alcohol extract of Qurs- i Bawāsīr

 

Figure 2: Peak list and Rf values for Ethyl acetate extract at 366nm wavelength

 

Figure 3: Peak list and Rf values for Ethyl acetate extract at 254 nm wavelength

Figure 4: Powder microscopy

 

 

DISCUSSION

The traditional system of medicine, including the globally recognized Unani system, holds significant popularity and serves a pivotal role in meeting the healthcare needs of populations worldwide. According to estimates by the World Health Organization (WHO), a substantial 80% of the global population relies on herbal medicine. The Unani system of medicine stands out by offering remedies for a diverse range of disorders, each having undergone rigorous clinical testing and validation over centuries. Notably, substances that may be inherently toxic        in their crude forms undergo meticulous processing and sublimation before their application. The recent surge in the popularity of various traditional medicine disciplines, including Unani medicine, highlights the need for developing standard operating procedures (SOPs) and standardizing different Unani formulations. This emphasis on standardization aims to ensure and enhance the acceptability and reliability of Unani medicine in contemporary healthcare practices. Against this backdrop, the present study was conducted to scrutinize a polyherbal Unani formulation known as Qurṣ-i Bawāsīr ^{14, 15, 16}.

The comprehensive analysis of the physicochemical parameters for the study formulation, Qurṣ-i Bawāsīr (QB), encompassed a detailed examination of organoleptic properties such as appearance, colour, odour, taste, and texture. Microscopic studies were conducted, and various tests, including the friability test, hardness test, disintegration time assessment, uniformity of weight, and loss of weight on drying at 105 ⁰C, were performed. Furthermore, the study delved     into parameters such as alcohol-soluble matter, water-soluble matter, hexane-soluble matter, total ash values, acid-insoluble ash values, pH values of 1% and 5% suspensions, High-Performance Thin Layer Chromatography (HPTLC), microbial load contamination, aflatoxins contamination, as well as the determination of heavy metals and pesticide residues. This multifaceted approach aimed to provide a thorough understanding of the formulation's composition and quality, ensuring a robust assessment of its safety and efficacy. The study formulation QB showed flattened disc shape in appearance, blackish brown in colour due to presence of Rasaut, the odour was found to be characteristic, bitter in taste due to presence of Maghz-i Neem (Neem seeds) and Reetha and the texture of QB was found to be slightly rough.

The microscopic evaluation of powder of QB was carried out and diagnostic characters of the individual ingredients present in the formulation were assessed. The powder of QB was found with the following character that is indicating the presence of genuine ingredients such as five to eight layers of cork cells are present below the cortex. Tracheids, thin-walled xylem fibers, crystals of calcium oxalate, sclereids, epidermal cells and stone cells were observed.

Friability of tablets is an important measure of tablet strength. Tablet tends to powder, chip and fragments when handled with lack of elegance. Conventional compressed tablets that lose 0.5 to 1% of their weight are generally considered acceptable. The mean percentage of friability of QB was found to be in the range of 0.05 to 0.06% among three different batches of the formulation.

Tablet hardness test means, it is the force required to break the tablet in a diametric compression test. Very hard tablets will have a longer disintegration time that will ultimately slow down the dissolution process and subsequently impaired absorption of the drug while too soft tablets will not be able to withstand the mechanical pressure of packaging, shipping and transport¹⁷. The mean percentage of hardness of QB was found in the range of 8 to 9 kg/cm² in three different batches which is found within the limit.

According to USP (United States Pharmacopoeia) uncoated tablets have disintegration time standards 5 to 30 minutes when placed in a liquid medium^{18, 19, 20}. The mean value of disintegration time of QB in aqueous media was found in around of 35 minutes in three different batches which is slightly increases in comparison to the maximum permissible limit of 30 minutes.

The weight of the tablet supports to maintain the efficacy of that tablet and contains the proper amount of drug. The tablets meet the test (more than 324 mg category); if no more than two tablets are outside the percentage limit of 5% ²¹. The mean value of randomly selected 20 tablets was found to be 546 ± 9.46 mg. The deviation from the average weight of each tablet was found within percentage limit of 5%.

Loss of weight on drying (LOD) at 105 ⁰C indicates the amount of water content as well as volatile components present in a particular sample. It helps to reduce errors in the estimation of the actual weight of drug material. Low moisture content in the drug suggests better stability against degradation and decomposition of drug or product either due to chemical change or microbial contamination, that ensure the quality and stability of drug²². Normal range of LOD is up to 14% in single drugs^{23, 24}. The mean percentage of loss of weight on drying at 105 °C QB was found to be in the range of 4.200-4.400 % w/w in three different batches that is within the limit.

For QB extractive values were found in the range 20.4893-21.2567, 64.9811-67.2113 and 8.2438-10.0953 % w/w in three different batches respectively.

The determination of the ash value provides a criterion for judging the identity and purity of the drug ²⁵. The mean percentage values of the total ash and acid insoluble ash for QB were found in the range of 12.0024-12.3569 and 5.0511-5.3262 % respectively.

pH is an important parameter of standardization which affects the absorption, distribution, metabolism, excretion, toxicity, permeability and bioavailability of drugs under varying pH conditions. Generally, drugs need a suitable degree of lipophilicity to reach the site of action, commencing with absorption from the GI tract, and interact with the appropriate receptor. Hence, the drugs which are weak acidic are better absorbed from the stomach than from the upper intestine^{26, 27}. The mean percentage pH of 1% and 5% aqueous suspension of QB were found in the range of 5.96-5.98 and 5.28-5.33 % w/w in three different batches respectively and both are weakly acidic.

In present study, a systemic phytochemical screening was carried out in different solvents (aqueous, methanol and hexane) extracts of the QB and the qualitative test was done and reported the nature of phytoconstituents present in the formulation.

In this study the HPTLC studies of ethyl acetate extract of QB was performed for the separation of different compounds present in the solvent extract and Rf values of various spots appeared in the TLC plate. The HPTLC of ethyl acetate extract of QB was evaluated with solvent system toluene: ethyl acetate: methanol (7:2:1, v/v/v) as the mobile phase. The TLC plate showed eight major spots under UV 366nm at Rf values 0.24,0.35,0.39,0.43,0.52,0.64,0.73,0.97 and under UV254nm showed ten spots at Rf values 0.27,0.32,0.36,0.41,0.50,0.57,0.65,0.71,0.78,0.95. HPTLC technique is also widely used for identification, detection of adulterants and helps in quality control. Advantage of HPTLC is that the several samples can be run simultaneously by use of a smaller quantity of mobile phase and HPTLC can investigate for simultaneous assay of several components in a multi-component formulation [14]. Moreover, the HPTLC analysis was studied using the same detection system and the mobile phases and the corresponding densitograms were recorded in which different peak observed for the spots appeared in the TLC plate. Each peak area was recorded using the HPTLC instrument. Thus, HPTLC technique is very crucial and important to detect the number of components in the extract and can provide quantitative aspects as well using the peak areas recorded in the densitogram obtained.

Safety evaluation of QB was done in which microbial load, aflatoxin, heavy metals and pesticide residue was evaluated and the study formulation QB is found to be in the range of 14X10²/g, 12 X10²/g and 13 X10²/g which are found within the permissible limit as per UPI²⁸ and WHO guideline while Salmonella spp., E. coli and total Yeast & Molds count were  found to be nil (table 3, 4). Therefore, prepared formulation QB is safe for consumption.

Aflatoxins includes under safety evaluation for the mycotoxins presence, which were evaluated through Aflatoxins standards using B1, B2, G1, and G2. The results for Aflatoxins contamination is found nil (table 4). Hence, it is indicated that the drug QB is safe and free from any mycotoxins or in particular Aflatoxin’s contaminations in the prepared formulation of all three               different batches.

Contamination through heavy metals such as lead, copper, arsenic, mercury, and cadmium in herbal medicine can be attributed to many causes, including accidental & environmental pollution and can cause clinically relevant dangers for the health of the user and should therefore be within the limit as prescribed by WHO & UPI²⁸. The Heavy metals Lead (Pb), Cadmium (Cd), Arsenic (As) and Mercury (Hg) were examined in the study formulation and they were not detected during the assay.

The assessment of pesticide residue was conducted at Bureau Veritas India Testing Services Private Limited, situated in the Industrial Estate Sanath Nagar, Hyderabad, Telangana. The results of this analysis indicated that the levels of pesticide residue were below the limit of quantification. Therefore, based on the findings presented in the table, it can be concluded that the formulated product QB is deemed safe for consumption, affirming its quality and adherence to established safety standards.

Upon careful consideration of the aforementioned discussions, it can be deduced that the quality control parameters assessed for the study formulation (QB) align with the guidelines provided by reputable organizations such as the (WHO) or other regulatory bodies. These meticulously evaluated quality control parameters serve as a benchmark, offering a reference point for certifying the authenticity and quality of the drug. This certification is vital in ensuring the continued efficacy and safety of the drug, thereby upholding the standards set forth by regulatory authorities.

CONCLUSION

In the present study, the Polyherbal Unani formulation Qurṣ-i Bawāsīr was subjected to the development of pharmacopeial standards using modern analytical techniques, which is crucial for establishing these standards. The data generated by the study could be used to set limits for quality control purposes, thereby achieving the maximum safety and efficacy of the QB. Further clinical studies can be designed to explore a more research-oriented perspective of the study formulation. Thus, this research confirms the quality standard of the Polyherbal Unani formulation Qurṣ-i Bawāsīr.

Acknowledgements: The authors are grateful to the In-Charge Director of the National Research Institute of Unani Medicine for Skin Disorders, Hyderabad, for providing the necessary facilities to conduct the study. 

Conflict of Interest: he authors declare that they have no conflicts of interest.

Author Contributions: All authors have equal contributions in the preparation of the manuscript and compilation.

Source of Support: Nil

Funding: The authors declared that this study has received no financial support.

Informed Consent Statement: Not applicable. 

Data Availability Statement: The data presented in this study are available on request from the corresponding author. 

Ethical approval: Not applicable.

 

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