Physicochemical Standardization of Majoon Hajrul Yahood: An important Pharmacopeial Unani Formulation

Introduction

In the recent times, there has been a shift in the preference of medicines from synthetic to herbal, which can be termed as “return to nature”. Medicinal plants have been known worldwide to a rich source of therapeutic agents for the prevention and treatment of various ailments of the body¹ . The Unani system of medicine uses plant-, animal-, and mineral-origin drugs, alone or in combination, in different dosage forms like Majoon (confection), Sufuf (powder), Joshanda (decoction), Qurs (tablet), Kushta (calx), Sharbat (syrup), and Arq (distilled). Individual natural drugs and their products have been comprehensively documented in traditional, as well as scientific literature for their healing properties^2,3. The secondary formulations are prepared using these primary preparations, to increase their efficacy, shelf life, acceptability (palatability), etc. Majoon Hajrul yahood is one such formulation that is frequently recommended for the treatment of urological disorders⁴ . Drug combinations often present promising results in the management of various diseases, compared to a single drug. This concept is well established in Western medicine, and remarkable success has been achieved over the decades. In the recent times, drug combination therapies for cancer and infectious diseases have offered a new hope to patients. Natural herbs and herbal ingredients organized in different formulations have shown potential interaction effects such as mutual enhancement, assistance, restraint, and antagonism⁵ . There has been a huge demand for plant-derived products in developed countries and are being increasingly sought for preparing medicinal products, nutraceuticals, and cosmetics⁶.

Single and combination herbal drugs contain a myriad of compounds in complex matrices in which no single chemical constituent is responsible for the overall efficacy. This creates a challenge in establishing quality control standards for raw materials and the standardization of the final herbal drugs thus obtained⁷ . Standardization assures a consistently stronger product with guaranteed constituents. Polyherbal formulations have received widespread acceptability as therapeutic agents for several ailments⁸ . Majoon Hajrul yahood is a herbo-mineral formulation extensively used in the Unani system of medicine for the management of urolithiasis. There is growing public interest in this herbal medicine, partly due to the limited number of options in pharmacotherapy particularly for the treatment of urolithiasis, which needs both preventive and curative remedy. Previous preclinical studies and clinical trials reveal that phytotherapeutic agents could be useful as either an alternative or an adjunctive therapy in the management of urolithiasis^9,10. Majoon Hajrul yahood contains Habbe Kaknaj (Physalis alkekengi L.), Hajrul yahood (Lapis judaicus), Maghz Tukhm Kharbuza (Cucumis melo L.), Maghz Tukhm Khayar (Cucumis sativus L.), and Maghz Kaddu (Lagenaria vulgaris) ^11,2. The primary aim of the present research was to standardize the formulation.

Materials and Methods

Raw materials

The ingredients for preparing Majoon Hajrul yahood were purchased from an authentic herb supplier in the local market of Bangalore, India. They were identified at the Trans-Disciplinary University (TDU), Bangalore, by Dr. S. Noorunnisa Begum, Associate Professor, (FRLHT Acc. No. 5042, 5043, 5044 and 5045). Lapis judaicus was identified and characterized by the Regional Ore Dressing Laboratory, Bengaluru, the Indian Bureau of Mines (IBM), Ministry of Mines Government of India, (report investigation No. K-23011/4/Chem/2018-19/ Analys/Bng/OD). A voucher specimen (Ref. No. 58/ IA/Res/2019) was deposited in the Department of Ilmul Advia, drug museum, NIUM, Bangalore, for future reference. In-house preparation of Majoon was done as per the method described in the classical book^11,2.

Quality control parameters of formulation

Organoleptic characteristics like smell, color, odor, taste, and consistency were evaluated. Physicochemical analysis of the formulation was carried out to evaluate the extractive values, pH, water- and alcohol-soluble contents, ash values (total ash, water-soluble and acidinsoluble ash), and loss of weight on drying. The pH of the various formulations was determined by using a digital pH meter (digital pH meter 7007, Digisun electronics, Hyderabad), and the moisture content was analyzed with Wensar moisture analyzer (ACZET series, Sl. No.:72521)^3,12-16.

Phytochemical evaluation of Majoon Hajrul yahood

The formulation was evaluated for alkaloids, carbohydrates, saponins, phenols, glycosides, phytosterols, flavonoids, proteins, and amino acids¹⁷.

Analysis for microbial contamination, pathogens, and aflatoxins

The culture tests comprising colony count analyses for the following microbes/pathogens were performed to test for pathogenic microbial contamination in the formulation:

Bacteria

Majoon sample 10 mg was dissolved in 1 mL of sterile saline (0.8% sodium chloride [NaCl] w/v). Luria-Bertani (LB) agar media containing Tryptone 10 g, NaCl 10 g, yeast extract 6 g, agar 15 g, distilled water 1000 mL was prepared and autoclaved at 121°C for 15 min. The sample (100 µL) was poured into sterilized petri plates and spread thoroughly. Approximately, 25 mL of the agar media was poured into the plate, allowed for solidification (pour plate method), and incubated at 37 °C. The cultures were studied after 24 h.

Fungi

Majoon sample (10 mg) was dissolved in 1 mL of sterile saline (0.8% NaCl w/v). Potato dextrose agar (PDA) media consisting of potato 200 g boiled in 500 mL distilled water and filtered, dextrose 20 g, agar 20 g, and distilled water 1000 mL was prepared. The remaining component was added to the filtrate and the volume was made up to 1000 mL with distilled water, and autoclaved at 121°C for 15 min. The petri plates were prepared for the culture as described above, and the plates were studied after 72 h.

Staphylococcus aureus

Mannitol salt agar (MSA) media contains beef extract 1 g, peptone 10 g, NaCl 75 g, D-mannitol 10 g, agar 15 g, phenol red 0.025 g, and distilled water 1000 mL. It was prepared at pH 7.4 and autoclaved at 121°C for 15 min. The petri plates were prepared for the culture as described above, and the cultures were studied after 24 h.

Pseudomonas

Cetyl trimethylammonium bromide (CTAB) media containing glucose 20 g, peptone 10 g, beef extract 1 g, CTAB 0.78 g, methylene blue 0.002 g, yeast extract 0.5 g, agar 17 g, and distilled water 1000 mL was prepared at pH 7.4 and autoclaved at 121 °C for 15 min. The petri plates were prepared for the culture as described above, and the cultures were studied after 24 h.

E. coli

Eosin-methylene blue (EMB) media contains peptone 10 g, lactose 5 g, di-potassium hydrogen phosphate 2 g, eosin 0.4 g, methylene blue 0.065 g, agar 15 g, and distilled water 1000 mL was prepared by setting pH 7.4 and autoclaved at 121°C for 15 min. The petri plates were prepared for the culture as described above, and the cultures were studied after 24 h.

Salmonella

Xylose lysine deoxycholate agar (XLD) media contains Yeast extract 3 g, L-lysine 5 g, Xylose 3.75 g, Lactose 7.5 g, Sucrose 7.5 g, Sodium deoxycholate 1 g, Sodium chloride 5 g, Sodium thiosulphate 6.8 g, Ferric ammonium citrate 0.8 g, phenol red 0.08 g, Agar 20 g, Distil water 1000 mL was prepared by setting pH 7.4 and autoclaved at 121°C for 15 min. The petri plates were prepared for the culture as described above, and the cultures were studied after 24 h.

Estimation of Flavonoids

The total flavonoid content was measured by the aluminium chloride colorimetric assay. Different aliquots of standard solutions of rutin (40, 80, 120, 160, and 200 μg/mL) were added to 10 mL volumetric flasks containing 4 mL of distilled water. To this, 0.3 mL 5% NaNO2 was added followed by the addition of 0.3 mL 10% AlCl3 after 5 min. Then, 2 mL 1 M NaOH was added after 5 min, and the volume was made up to 10 mL with distilled water. The solution was mixed, and absorbance was measured against the blank at 510 nm. The samples were processed in a similar manner by taking 100 µL of the sample (100 mg/mL). The total flavonoid content was expressed as mg rutin equivalents (RE)^[18,19].

Estimation of Tannins

Different aliquots of standard gallic acid (200 µg/mL) were poured into test tubes and 7.5 mL of distilled water was added to all the test tubes. Later, 0.5 mL of FolinCalcioteau reagent and 1 mL of 35% sodium carbonate were added in all the tubes. The volume in each test tube was made up to 10 mL with distilled water, and the absorbance was read at 725 nm. The samples were also processed in a similar manner by taking 100 µL (100 mg/mL) of the sample. The standard graph was plotted, and the amount of carbohydrates in each sample was calculated.

Estimation of Saponins

Different aliquots of standard saponin (1 mg/mL) were taken in test tubes, and the volume was made up to 1 mL with absolute methanol in all the test tubes. Later, 500 µL of 8% vanillin and 500 µL of 72% sulphuric acid were added in all the tubes and incubated at 60 ºC for 10 min. The absorbance was read at 544 nm. The samples were also processed similarly by taking 100 µL (100 mg/ mL) of the sample. The standard graph was plotted, and the amount of saponin in each sample was calculated.

Heavy metal contamination estimation

A 0.5 g of sample (wet weight) was taken in a vessel and 10 mL of conc. HNO3 was added. The samples were allowed to pre-digest by standing open for 15 min before sealing the vessels. These vessels were then kept in a microwave digester (Model: Mars 6). After the digestion, the vessels were allowed to cool and the samples filtered and made up to 25 mL with water. The above solution was aspirated and analyzed using inductively coupled plasma-optical emission spectrometry (ICP-OES; Teledyne Leeman Labs, Model: Prodigy 7).

HPLC analyses

Flavonoids: High-performance liquid chromatographic (HPLC) analysis was done in an isocratic mode with mobile-phase acetonitrile and water in the ratio 7:3 with the RP-HPLC C-18 column at a flow rate of 1 mL/min. The standard quercetin (0.4 mg/mL) and samples (10 mg/mL) were dissolved in mobile phase and 20 µL was injected, and the elution was monitored at 272 nm.

Saponins: The process used for analyzing flavonoids, mentioned above, was followed for the analysis of saponins, except the acetonitrile-to-water ratio was 4:6 and the standard used was saponin (0.4 mg/mL). The elution was monitored at 203 nm.

Tannins: Mobile-phase methanol and water in the ratio of 1:1 with the RP-HPLC C-18 column at a flow rate of 1 mL/min were used for this analysis. The standard gallic acid (0.4 mg/mL) and samples (10 mg/mL) were dissolved in mobile phase and 20 µL was injected, and the elution was monitored at 270 nm.

Results

Organoleptic characteristics

The Majoon Hajrul yahood prepared in-house was found to be semisolid in consistency with yellowish-brown color. It had a characteristic sweetish smell and taste.

Physicochemical analysis

The formulation was noted to have a total ash content of 10.6% w/w, acid-insoluble ash of 1.5% w/w, and watersoluble ash of 6% w/w. The pH of 1% and 10% solutions was of 6.29 and 5.82, respectively.

Phytochemical analysis

Preliminary phytochemical analysis of the aqueous extracts of Majoon Hajrul yahood’s ingredients showed the presence of alkaloids, carbohydrates, saponins, phenols, glycosides, phytosterols, flavonoids, proteins, and amino acids.

Discussion

Herbal products have been extensively used over the years in the management of various diseases. Majoon Hajrul yahood which is a multifaceted drug widely used in the Unani system of medicine has been analyzed in the present work. An in-house formulation was prepared in accordance with the national formulary of Unani medicine (NFUM). Standardization of Majoon Hajrul yahood performed following the guidelines of NFUM and the protocols mentioned in the Ayurveda, Sidhha and Unani systems^16,20. The finished product, Majoon Hajrul yahood, was tested for relevant physical and chemical parameters. The organoleptic properties such as smell, color, odor, taste, and consistency of the food and other substances are experienced by the senses. Any change in these properties gives a primary indication about quality variations and might be useful for distinguishing it from its substitutes and adulterants³ . Established preliminary and physicochemical standards give important information for further investigations and facilitate the identification of formulations. The test for percentage of moisture content determines both the water and volatile matter. The total volatile and moisture content of the drug was established by its loss on drying. The moisture content of the drug reveals its stability and shelf life. A high moisture content may adversely affect the active ingredient of the drug and cause early contamination of the drug. Low moisture content could provide the maximum stability and a better shelf life ¹⁵. The moisture content of the drug in our study was 0.26%. Iqbal et al reported 12.1% moisture content for nonpharmacopeial formulation. Moisture content ranging from 10-20% has been reported to be ideal for the growth of different types of microorganisms¹⁴.

The ash value determination forms the basis for judging the identity and cleanliness of a drug and provides information related to its adulteration by inorganic matter²¹. The total ash content indicates the amount of materials remaining after ignition whereas acidinsoluble ash is the amount of silica present after ignition, especially sand and siliceous matter.

Extractive values are useful for evaluating the consistency and amount of chemical constituents present in a drug. In the present study, Majoon Hajrul yahood was also evaluated for all these parameters. The extractive values, such as water- and alcohol-solubility, indicate the amount of the active constituent and the bioavailability of the plant. A lower value indicates the presence of the exhausted material. In the present study, the in-house preparation had maximum aqueous-soluble extractive value than hydroalcoholic-soluble extractive values²². The pH is useful for quantitative indication of the acidity and alkalinity of a solution. The present formulation had a pH of 6 indicating that it is acidic in nature. In a study conducted by Iqbal et al, the formulation had a pH of 6.30 and 9.28 for 1% and 10% solutions, respectively²¹. Abdullah et al found that for Majoon-e-Piyaz, these values were 4.77 and 4.88, respectively. A study on Majoon-e-Aarad khurma performed by Mujeeb et al reported that the pH of this formulation was 4.3 and 7.2, respectively²².

Phytochemicals are naturally present in plants and may vary from plant to plant and also between different samples of the same species, depending upon various atmospheric factors, storage, and drying conditions. A slight deviation from the normal in terms of quality and quantity may alter the effect of the drug. Apart from the degradation in the quality of the drugs that occurs due to the above conditions, adulteration also contributes to its variability¹⁴. Majoon was also evaluated for the presence of phytochemicals and was noted to contain alkaloids, carbohydrates, saponins, phenols, glycosides, phytosterols, flavonoids, proteins, and amino acids.

Previous studies reported that the plant extract which is rich in tannins and flavonoids can lead to relaxation of the smooth muscles of the urinary tract which could facilitate the expulsion of stones from the kidney and diminish the size of calculi in rats9 . Flavonoids have been reported to inhibit calcium oxalate crystallization, and saponins are known to possess anticrystallization properties²³.

Due to their origin, herbal drugs are prone to contamination by microorganisms from soil, air, and water which may be potentially pathogenic to man. The presence of microbial contaminants in herbal products can reduce or even inactivate the therapeutic potential of the products and can adversely affect patients taking these medicines. Therefore, manufacturers should ensure the lowest possible level of microorganisms in the raw material and finished dosage forms to maintain appropriate quality, safety, and efficacy of the natural products. The microbial load in Majoon Hajrul yahood was found to be within the limit mentioned in the Unani pharmacopeia of India16. In this formulation, the total bacterial and fungal counts were 66 and 10, respectively. Salmonella, Pseudomonas, Staphylococcus, and E. coli were absent, indicating the microbiological safety of the formulation for human use. Classes of compounds such as alkaloids, saponins, tannins, etc., have demonstrated activity against several pathogens and therefore, are traditionally used for the treatment of various illnesses ¹⁸

The heavy toxic metals contamination in plants could develop serious health problems as there is a narrow concentration range between the deficiency and toxicity levels of heavy metals in humans. The WHO has emphasized on various standard techniques for the analysis of toxic heavy metals in plant products to ascertain their safety. Heavy metals were analyzed by ICP-OES, and it was found that cadmium and mercury were below the detectable limit; and lead and arsenic was within the permissible limit¹⁶, ensuring the safety of Majoon Hajrul yahood for human consumption. Inorganic constituents like calcium, magnesium, and zinc were also analyzed and found to be present in the formulation. Aflatoxins, the secondary metabolites produced by the Aspergillus species, contaminate a variety of agricultural and food commodities. They are classified into a number of subtypes; however, the most important ones are B1, B2, G1, and G2. These mycotoxins are recognized to be hepatotoxic and carcinogenic for humans. The formulation was tested for aflatoxins (B1 , B2 , G1 , and G2 ) by the aflacord method and was found to be <5.0.

The natural plant products can also act against pathogenic bacteria in humans. Staphylococcus aureus is a major human pathogen that is capable of causing persistent skin infections²⁴. Secondary metabolites such as phenols, alkaloids, glycosides, quinones, essential oils, tannins, saponins, flavonoids, etc., present in the extract inhibit bacterial respiration and intensify plasma membrane permeability, causing death of bacterial cells²⁵. As far as antimicrobial activity of the formulation is concerned, the flavonoid and phenolic content exhibited inhibitory effects against multiple microorganisms. The antimicrobial action of polyherbal formulations is a result of the interaction between the selected herbs and the cell membrane of the target microorganisms, which probably binds with extracellular and soluble proteins and also with cell walls. The resultant membrane disruption changes the structure and function of key cellular constituents, resulting in mutation, cell damage, and death²⁶.

Conclusion

Scientific evaluation of polyherbal formulations is vital. This study may serve as a standard reference for developing standard operating procedures for quality control analysis of Majoon Hajrul yahood.

Abbreviations

ICP-OES, Inductively Couple Plasma-Optical Emission Spectrometry; NFUM, National formulary of Unani medicine; NIUM, National Institute of Unani Medicine; HPLC, High Performance Liquid Chromatography.

Conflict of interests

The authors declare no conflict of interests.

Acknowledgment

The authors are thankful to the ex-director of NIUM, Prof. Mohd. Zulkifle, and the present director, Prof. Abdul Wadud for providing the best possible facilities for carrying out the research. Authors also express their gratitude to the Indian Bureau of Mines, Regional Mineral Processing Laboratory & PP, Goruguntapalaya, Bangalore, for their help in the characterization of Hajrul yahood.