Determination of Quality Standards and Evaluation of In vitro Antibacterial Activity of Kanchanara Guggulu: An Ayurvedic Formulation

Abstract

Introduction: 

Ayurveda is one of the most renowned Indian traditional health-care systems and has been adopted globally in recent decades. In Ayurveda Kanchanara guggulu (KG) tablet is one of the Indian traditional medicines used in the treatment of various indications such as tonsilitis, cyst, abdominal lump, and fistula-in-ano.

Methods: 

In the present study, KG tablet was prepared and standardized on basis of physical, physico-chemical properties, high-performance thin-layer chromatography (HPTLC) fingerprint, and phytochemical analysis. Heavy metal test of lead, arsenic, mercury, and cadmium was carried out to confirm the permissible limit as per the AYUSH and WHO standards. The antibacterial activity of the KG tablet was evaluated on two human pathogenic bacteria Escherichia coli and Staphylococcus aureus.

Results: 

The heavy metal limit was found below the limit of quantification and within the allowable limit as per the AYUSH and WHO standards. The tablet showed comparable antimicrobial activity against both strains. The tablets were found to be having satisfactory quality parameters such as hardness, disintegration test, and friability values as per the pharmacopeia standards.

Conclusion: 

The tested parameters based on the present study can be used as a reference approach for quality control of KG formulation. The HPTLC study was found to be an effective tool for quality control of herbal formulation to check the inter-batch batch consistency of the formulation. The antimicrobial activity against human pathogens substantiates its traditional uses.

Introduction

Guggulu is a well-known medicine in Ayurveda having enormous uses. It is a pale yellow to a honey-brown-colored oleo-gum resin that exudes from the stem and branches of Commiphora mukul (syn Commiphora wightii) tree.^[1,2] Guggulu-based preparations are the important formulation in the Ayurveda used for a variety of therapeutic indications. The Kaisora guggulu, Gokshuradi guggulu, Triphala guggulu, Yogaraja guggulu, and Kanchanara guggulu (KG) are some of the important Guggulu-based formulations in Ayurveda.^[3] The KG is one of the classic ayurvedic medicines. It is used for various therapeutic indications such as gulma (abdominal lump), gandamala (cervical lymphadenitis), apaci (scrofula), granthi (cyst), vrana (ulcer), kustha (disease of skin), bhagandhra (fistula-in-ano), and slipada (filariasis).^[3,4] The formulation is a polyherbal medicine consisting of herbal ingredients Bauhinia variegata (stem bark), Terminalia chebula (fruit), Terminalia bellrica (fruit), Phyllanthus emblica (fruit), Zingiber officinale (rhizome), Piper nigrum (fruit), Piper longum (fruit), Crataeva nurvala (stem bark), Elettaria cardamomum (fruit), Cinnamomum zeylanicum (stem bark), Cinnamomum tamala (leaf), and Commiphora weigtii (oleo gum resin). Among them, Bauhinia variegata and Commiphora weigtii are the major ingredients that constitute 25% and 62%, respectively, in the formulation.^[5]

Materials and Methods

All the herbal ingredients [Figure 1] of KG tablet were procured from the different markets across India and were validated by a botanist in laboratory. The herbarium sample was stored for future references. The herbal ingredients were evaluated for pharmacopeial parameters as per the recommended methods. All the chemicals and reagents used were of analytical grade and were purchased from corresponding suppliers. The phytochemical standards of guggulusterone E and Z were purchased from natural remedies. All the excipients used in the tablet formulation were of pharmaceutical excipients grade. The media and broth used in the antimicrobial activity and microbial enumeration were purchased from HiMedia company.

Figure 1: Herbal drugs of Kanchanara guggulu tablet

Purification of guggulu oleo-gum resin (Guggulu suddha)

In Ayurvedic and Traditional Chinese Medicines principles, some of the herbs must be be purified before being used in the formulation. As per the traditional principles, these processing techniques detoxify and neutralize the toxic principles present in the drug and enhance the therapeutic effects of the drugs. The purification process includes frying, steaming, boiling, and processing with various liquids such as cow urine, milk, herbal juice, and herbal decoction.^[6] As the administration of raw guggulu reported to cause adverse reactions, for example, skin allergy, irregular menstruation, gastric irritancy, diarrhea, headache, and liver toxicity at higher doses. As per the principles of Ayurveda, Guggulu must be purified prior to use in the formulation.^[3] For purification of Guggulu, gomutra (cow urine), triphala kasaya (decoction from fruits of the Emblica officinalis (Amalaki), Terminalia bellirica (Bibhitaki), Terminalia chebula (Haritaki) in equal portion), vasapatra kasaya (decoction of Adhatoda vasica leaves), vasapatra savrasa (aqueous extract of Adhatoda vasica leaves), dugdha (milk), and water are used.^[3]

In this, foreign impurities such as sand, stone, and glass were removed during the purification process and guggulu gum was broken into small pieces and bundled in a cotton cloth followed by boiling with Triphala decoction. In this process the Guggulu become soft in consistency, passed through cotton cloth and mixed into the triphala decoction. The resulting mixed extract of guggulu and triphala decoction was filtered using 100 mm filter cloth and the resultant filtrate was collected and the residues in the cotton cloth were discarded. The fluid collected was then tray-dried under vacuum to get an oily, soft texture, and brown-colored sticky product called as Guggulu Suddha or Sodhit Guggulu.^[2,3]

Preparation of Kanchanara guggulu tablet

All the ingredients of pharmacopeial quality except the Guggulu were verified, washed, dried, pulverized, and passed through the sieve of mesh size 80 to get a fine powder. The resulting fine powder was mixed with the required quantity of purified Guggulu (suddha-guggulu) to get a homogenous mixture. The extract was converted into tablets by using the excipients maize starch-12%, micro crystalline starch –7.2%, sodium starch glycolate –2.1%, magnesium sterate –0.5%, and aerosol –3.5% by direct compression method. Furthermore, the tablets were subjected to aqueous-based film coating using hydroxypropyl methylcellulose based to protect from moisture.

Evaluation of tablets

Various physical properties such as disintegration, friability, hardness, weight variation, water-soluble extractive value, alcohol-soluble extractive value, and ash value were evaluated as per the Indian pharmacopoia methods.

Dimensions of the tablets

Tablet thickness and length were measured using a digital Vernier caliper (aerospcace, 0–150 mm).

Disintegration test

The time for the disintegration of tablet was measured using USP disintegration apparatus (Electrolab India Pvt Ltd., Model ED-2 L) in distilled water at 37°C ± 2°C.

Friability test

Friability study was carried out using Roche Friabilator (Campbell Electronics, India). Twenty accurately weighed tablets were placed in the Friabilator and rotated for 100 revolutions. The tablets were then dusted to remove any dust and reweighed. The percentage of weight loss was calculated as friability. According to the pharmacopeial specification, friability loss should be <1% without any crack, cleaved, or broken tablet observed after the test.

Hardness

Tablet hardness was measured by using the Monsanto hardness tester (Cadmach, India).

Weight variation

Twenty tablets were sampled randomly and the average weight was calculated. Shimadzu’s analytical balance (Model ATY-224) was used for weighing measurement.

FTIR analysis

A Shimadzu-FTIR-8400S FT-IR spectrometer was used for recording FTIR spectra of the active extract with KBr pellets. The scanning was done between 500 and 4000 cm⁻¹.

Microbial limit study

The analysis of microbial limits was studied as per the method mentioned in the Ayurvedic Pharmacopoeia of India (API).^[7] As per the API specification, Total Plate count (TPC) and total yeast and mold count (TYMC) should be <10⁵ and 10³ per gram of samples and Staphylococcus aureus, Salmonella Sp., Pseudomonas aeruginosa, and Escherichia coli should be absent per gram of samples. Various selective media were used for the identification and enumeration of microbial limits. Casein soya bean digest agar and broth were used for TPC and Casein soya bean digest broth and Sabouraud’s glucose agar with antibiotics were used for TYMC, Casein Soya bean digest broth and Cetrimide-agar for identification of P. aeruginosa, Casein Soya bean digest broth and Baird parker agar for S. aureus, Casein Soya bean digest broth and Brilliant green agar for Salmonella sp., and MacConkey’s broth and agar for the identification E. coli.

Phytochemical analysis

Preliminary phytochemical analysis of formulated tablet was performed for detection of various phytoconstituents such as alkaloids (Mayer’s test, Wagner’s test, Hager’s test), steroids (Dragendroff’s test), phytosterols (Liebermann-Burchard test), flavonoids (natural product polyethylene glycol reagent test, aluminum chloride test), phenolics (Ferric chloride test and lead acetate test), terpenoids (anisaldehyde sulphuric acid and vanillin sulphuric acid test), and carbohydrates (Molisch test and Fehling’s test) as per the standards reported methods. Physicochemical analyses of all the herbal raw materials were performed for quality control purposes.^[8–10]

High-performance thin-layer chromatography fingerprint of Kanchanara guggulu tablet

Precoated TLC plate with silica gel 60F254 of 0.2 mm thickness (Merck-Germany) was used for high-performance thin-layer chromatography (HPTLC) fingerprint. For HPTLC sample preparation, 5 g of coarsely powder of tablets and the raw materials were extracted with 50 ml of methanol by sonication. The alcohol-soluble fraction was filtered through the Whatman filter and the filtrate was used for HPTLC application. By using a Linomat V, applicator 8 mL of the sample was applied on the TLC plate with 8 mm band length using 100 mL Hamilton syringe. The plate was run in a twin trough glass chamber (CAMAG) using toluene: ethyl acetate: methanol: formic acid (7:2:1:0.5;v: V: V: V) as mobile phase. After the development, the plate was derivatized with anisaldehyde sulphuric acid and heated at 105°C for 5 min. The plate was visualized in CAMAG UV cabinet under 254 nm and 366 nm. CAMAG Scanner III equipped with winCATS software version 1.4.3 was used for scanning the plates. The TLC analysis was carried out with the standards of guggulsterone-E and Z for their identification in the tablet.

Antimicrobial activity

Pathogenic bacterial strains used in the study were Gram-negative bacteria (E. coli ATCC-8739™) and Gram-positive bacteria (S. aureus ATCC^® 6538™). For E. coli, MacConkey’s broth and MacConkey agar were used, while for S. aureus Casein Soya bean digest broth and Baird parker agar were used. Antibiotic test disc ciprofloxacin (CIP-30 mcg), azithromycin (AZM-15 mcg), and ampicillin-5 mcg (AMP-5 mcg) were used as a positive control. The agar well diffusion method was employed to determine the antibacterial activity KG tablets. The bacterial inoculum was spread over the nutrient agar plate using a sterile cotton swab to facilitate an uniform microbial growth. The holes of 6 mm diameter were made on the agar plates. For evaluation of the anti-microbial activity, 10g of the KG-tablet were extracted with ethyl alcohol and water and dried extract of was used for the study. The extract was dissolved in dimethyl sulfoxide at 100 mg/ml concentration. The antimicrobial activity was tested by adding 10 mg, 7.5 mg, 5 mg, and 2.5 mg of samples into each of the wells and the study was done in triplicate. The plates were incubated for 48 h at 37°C. The antimicrobial activity was evaluated by measuring the diameter of zone of inhibition (ZOI) in millimeters by antibiotic zone reading scale.

Heavy metal test of the formulation

The analytical measurement of Pb, Cd, Hg, and As in the digested sample were carried out with a PerkinElmer NexIon 2000 ICP-MS. The sample was digested in a microwave digester (PerkinElmer) with ultra-pure nitric acid procured from Honeywell research chemicals. The heavy metals analytical standards of Pb, Cd, Hg, and As were purchased from inorganic ventures.

Results

The formulated tablets were evaluated for tablet parameter, phytochemical, development of HPTLC fingerprint, and antimicrobial properties. All the quality control tablet parameters were in the range of acceptable limits [Table 1]. The friability of the tablet was found to be 0.16% which confirms the standards. The disintegration time was found to be 41 min. Comparatively high disintegration of tablet was due to the presence of Guggulu in the formulation. In general, the Guggulu tablets are reported to have disintegration time around 60 min. The microbiology parameters such as total aerobic count and total fungal count was within the limit as per the Indian Pharmacopeia standards. The pathogens E. Coli, S. aureus, P. aeruginosa, and Salmonella sp. were absent as per the pharmacopeia specifications.

Table 1: Results of pharmaceutical evaluation Kanchanara Guggulu tablet formulation

Antibacterial activity of Kanchanara guggulu

Antibacterial activity of alcoholic and aqueous extract of KG tablet were evaluated against pathogenic E. coli, S. aureus for antimicrobial activity [Table 2]. The alcoholic extract of the KG exhibited ZOI of 20 mm and 16 mm in S. aureus and E. coli, respectively, while aqueous extracts showed ZOI of 18 mm and 14 mm in S. aureus and E. coli, respectively, at a concentration of 10 mg/well. Although throat infection is majorly caused by viral infections, the visible antibacterial activity of the product may support the traditional use of KG tablets in treatment of throat infections.

Table 2: Antimicrobial activity of Kanchanara guggulu samples

High-performance thin-layer chromatography profiling of Kanchanara guggulu tablets

The HPTLC fingerprint profile of KG tablet along with its plant drugs at 254 nm, 366 nm, and 520 nm is presented in Figure 2 and Table 3. Twelve spots found in the KG tablet at 254 nm were due to the herbal ingredients used in the formulation. Out of 12 spots in KG, Rf values of 0.08, 0.20, 0.30, 0.45, 0.52, 0.61, 0.67, 0.75, and 0.86 were from track 7, 6, 3, 12, and 2. Spots in KG corresponding to Rf values of more than one ingredient would be due to the merging of more than one compound with same Rf values. A spot with an Rf value of 0.37 was observed in both track 1 and track 8; hence, the spot in KG corresponds to a mixture of a minimum of two compounds with the same Rf values. Similarly, the HPTLC R_f pattern at 366 nm and 520 nm for a finished product of KG tablets correlated with the corresponding raw materials. The E-guggulsterone and Z-guggulsterone were identified in the KG tablet at R_f of 0.12 and 0.23, respectively, after derivatization with anisaldehyde sulphuric acid as golden yellow color spots.

Figure 2: High-performance thin-layer chromatography fingerprint of raw materials and Kanchanara guggulu tablet

Table 3: R_f values of raw materials and Kanchanara guggulu tablet at 254 nm and 366 nm and under 520 nm postderivatization with AS reagent

Figure 3: FTIR spectra of the Kanchanara guggulu tablet

Preliminary phytochemical analysis

The phytochemical analysis of the KG extracts confirms the presence of alkaloids, carbohydrates, phenolics, phytosterols, and flavonoids in the aqueous and alcoholic extract of the tablet on the basis of various reagent-based tests. The physicochemical parameters are mentioned in below Table 4.

Table 4: Physicochemical parameters of herbs

FTIR (KBr) ν_max

The FTIR analysis of the herbal active extract showed the peaks at various wavenumbers corresponding to the several functional groups. The FTIR spectra displayed peaks at 3475 cm⁻¹ attributes to the vibration of hydroxyl (-OH) groups, the vibration at 2977 cm⁻¹, 2954 cm⁻¹, and 2876 cm⁻¹ were due to stretching vibration of = C-H,-CH2 groups, carbonyl group (C = O) stretching vibration was observed at 1745 cm⁻¹, peaks at 1622 cm⁻¹ and 1584 cm⁻¹ were due to aromatic stretching vibrations, the peaks at 1511, 1488, 1445, 1415, 1385, and 1307 cm⁻¹ were due to CH bending vibrations, the vibration peaks for C-O-C groups were observed at 1236, 1199, and 1132 cm^-1 and the peaks at 1084, 1035, 1014, 982, 904, 831 cm⁻¹ were due to CH bending vibrations. The observed hydroxyl, carbonyl, ether, alkene, alkane, and aromatic groups are the common functional groups found in various phytochemicals like phytosterols, phenolics, alkaloids, and flavonoids.

Heavy metal test results

The concentration levels of heavy metals Pb, Cd, Hg, and As (ppm) in the finished goods are presented in Table 5. Lead content in the sample was found to be 0.29 ppm and arsenic content was found to be 0.13 ppm. Cadmium and mercury were not detected in the product with a limit of quantification of 0.0125 ppm. The results of the heavy metals comply with the AYUSH limit as well the limit recommended by WHO in herbal drug quality control. As per the WHO monograph, permissible limit for lead is 10 ppm and cadmium is 0.3 ppm for herbal drugs and herbal formulations.^[11,12]

Table 5: Heavy metal limit of Kanchanara guggulu tablet from inductively coupled plasma mass spectrometry analysis

Discussion

The composition of tablet formulation was found to be optimum with acceptable physical parameters like hardness, friability, and disintegration time. Quality control of herbal medicines with polyherbal compositions is a difficult process due to the complex chemistry of the formulation. Therefore in the present research work, in order to standardize the tablet, an HPTLC method was developed for simultaneous analysis of herbal ingredients along with the finished formulation. The mobile phase consisted of toluene: ethyl acetate: methanol: formic acid (7:2:1:0.5; v: V: V: V) was helped in the optimum resolution of phytoconstituents within a shorter time. Guggulsterone E and Z were detected in the formulation so can be used as a chemical marker for standardization of the formulation. The extracts of the tablet showed moderate antimicrobial activity against the pathogenic bacterial E. coli and S. aureus. With the emergence of new infectious diseases of novel viral and bacterial origin, the consumption rate of antibiotic medicines has been increased by around 39% times in the last few decades. The defined daily dose has also increased by around 65%. Therefore, indiscriminate use of antibiotics has led to antibiotic resistance superbug. This is a serious concern as the resistant pathogens could be dangerous, kill, and get spread leading to enormous cost on global health costs. As the herbal drugs from Ayurveda are novel sources of the various antimicrobial agents due to their complex phytochemistry, they can be used for screening to identify the potential anti-microbial agent.^[13,14] Our study reports in vitro antibacterial activity of the drug kanchanara guggulu. With further clinical trial of the drug on human volunteers for its efficacy, it could be used as a drug candidate for the treatment of microbial infections.

Conclusion

The HPTLC fingerprint was found to be a promising tool that confirms the authenticity of the formulation indicating the presence of all the used herbal raw material in the KG tablet. The HPTLC fingerprint can be used for routine monitoring of herbal raw materials and finished products for batch-to-batch consistency before the product is released for the market. All the heavy metal tested were within the permissible limit as per the AYUSH and WHO standards. The tablets showed good antimicrobial activity on two tested pathogenic bacteria substantiating its traditional usage in infective conditions. As the majority of throat infections are of viral origin, further in vitro antiviral studies and clinical trials are needed for KG tablet to strengthen its traditional claims.

Financial support and sponsorship

This was a self-financing study.

Conflicts of interest

There are no conflicts of interest.