Pharmacognostic Evaluation and HPTLC Analysis of Multiple Phytoconstituents in Anoral Antidiabetic Polyherbal Ayurvedic Formulation

INTRODUCTION

‘Ayurveda’ one of the ancient sciences of medicine, originated in India and followed in many parts of India and other parts of the world, offers cure to various ailments in a holistic manner. Even today most of the chronic diseases which don’t have cure in any other system of medicine has cure in Ayurveda. The main advantage of ayurvedic system of medicine over conventional system of medicine (allopathic system) is that they do not have any kind of side effects when used in proper manner. In allopathic system, many chronic diseases are treated by prescribing long term medications after which patients have to generally suffer with many side effects.^1-3

Diabetes is one such disease where lifelong medication is a must to control blood sugar level at normal levels. Type II- Diabetes is a chronic disease condition in which pancreas fails to secrete sufficient insulin required for the body to keep blood glucose within limit or even though insulin is secreted, not used effectively by the body. In both of these conditions where there is reduced secretion or ineffective utilization of insulin, blood glucose will not enter the cells and as a result, sugar concentration increases in blood. This leads to increased sugar level (or glucose) in the blood (hyperglycemia). Once sugar level increases, a part of glucose gets infiltrated into urine and the condition is called as glucosuria. Diabetes is a life style disorder and some of the causes for diabetes are Obesity, Heredity, sedentary life style, Stress, Women with prior gestational diabetes, and Strain.^4-6 In Ayurveda, for the treatment of diabetes various herbal preparations are suggested.

Madhuniyantrak is an oral polyherbal formulation in the form of tablets manufactured by Karnataka Antibiotics Private Ltd, Bangalore (KAPL). It comprises of extract of twelve ingredients Viz., Gymnema sylvestre, Curcuma longa, Azadiracta indica, Momordica charantia, Trigonella foenum graecum, Aegle marmelos, Tribulus terrestris, Emblica officinalis, Terminalia chebula, Terminalia bellerica, Pterocarpus marsupium and Shuddha shilajit. ⁷ The formulation is prescribed by many physicians for management of Diabetes. Literature survey also supports the use of all the crude drugs incorporated in the formulation in controlling blood glucose levels in the body.

Even though the product has shown clinical efficacy and has been in the market, there are very few reports on estimation of constituents in the formulations. As per WHO and most of the regulatory authorities and also to have a better market, it is very important to have an analytical method for identification and determination of chief constituents in the formulation as a mark of quality check.⁸

In this study we make an attempt to establish an optimized method for simultaneous identification of five most important constituents of the formulation i.e., Curcumin, Emodin, Gallic acid, Ellagic acid and Ascorbic acid by HPTLC.

MATERIALS AND METHODS:

Materials:

• Madhuniyantrak tablets- obtained from KAPL, Bangalore.

• Standards extracts of all the herbal drugs incorporated in the formulation, Procured from KAPL.

• Crude drugs: Dried fruits of Tribulus terrestris, Emblica officinalis, Terminalia chebula and Terminalia bellerica (obtained from KAPL, Bangalore)

Leaves of Gymnema sylvestre, Rhizomes of Curcuma longa, leaves of Azadiracta indica, fruits of Momordica charantia, and seeds of Trigonella foenum graecum, Leaves of Aegle marmelos, Dried Heart wood of Pterocarpus marsupium and Shuddha shilajit (obtained from local market in Bangalore).

Identification and authentication of plant material was done at Ayurveda Dietetics Research Institute, Jayanagar (Bangalore). Shilajit identification and authentication was done by Dr. K. Madhava chetty, Assistant professor, department of Botany. Sri Venkateshwara university, Tirupati.

Methods:

The purpose of the study is to identify the presence of Curcumin, Emodin, Gallic acid, Ellagic acid and Ascorbic acids in the (i) crude drugs incorporated in the formulations (ii) Extracts used for manufacture of the formulation and (iii) Formulation- Madhuniyantrak oral tablets.

To achieve this identification is done for presence the marker compounds

• In the each of the extracts, extracted by using the crude drugs in our laboratory by TLC.

• In the mixture (prepared in the ratio as given in the formulation) of extracts, extracted using crude drugs in our laboratory.

• In the mixture (Prepared in the ratio as given in the formulation) of ready extracts obtained from KAPL which are actually incorporated in the formulation.

• In the formulation –Madhuniyantrak (Oral polyherbal tablet obtained from KAPL, Bangalore)

With this it’s been attempted to prove the presence of marker compounds in crude drugs, extracts incorporated in the manufacture of formulation and the formulation itself.

Organoleptic and proximate analysis for the crude drugs and the extracts.

All the crude drugs were observed for organoleptic characters such as color, odor, size, shape, texture, form etc.

Proximate analysis:

All the crude drugs were analyzed for parameters such as Moisture content, Extractive values (Methanol) and ash values as a part of standardization process as per WHO guidelines.

Extraction of Crude drugs:

Selected plant materials were air dried at room temperature, powdered in blender and passed through sieve No 10. The sieved powders of Curcuma longa, Momordica charantia,Trigonella foenum graecum, Tribulus terrestris, Emblica officinalis, Terminalia chebula, Terminalia bellerica Pterocarpus marsupium and Shuddha shilajit was weighed accurately andsubjected separately to extraction by using methanol as a solvent in Soxhlet apparatus.

Gymnema sylvestre, Azadiracta indica and Aegle marmelos were defatted using petroleum ether, thenthe powder material was removed and air dried to remove the adhering solvent and extracted with methanol. All the drugs were extracted at 60-80 °C temperature till the completion of the extraction. Extract obtained was filtered and concentrated on hot plate. Percentage yield of extract was calculated. Dried extracts were labelled and stored in air tight containers for further studies.⁹

Evaluation of Quality Control Parameters for selected medicinal plants: Extractswere analyzed for organoleptic properties and proximate analysis. Preliminary phytochemical analysis was performed as per standard procedures.^10-13

Standardization of extracts as per WHO guidelines: Standardization of extracts was carried out as per WHO guidelines with respect to limit test for arsenic, nickel, cadmium and lead and microbial content.

High Performance Thin Layer Chromatography:

Optimization of mobile phase solvent system:

Various solvent systems were tried with different ratios for proper separation of phytoconstituents for each of the extracts. Solvent system which clearly separated maximum constituents was selected.

Identification of marker compounds by HPTLC

HPTLC is one of the best suited techniques available for identification of constituents from plant extracts. This technique is highly accurate and precise with high flexibility in different stages of experimental steps (Mobile phase, stationary phase, development process and detection). Purity of the crude drugs and identification of specific phytoconstituents in the raw herbal extracts and finished formulations can be effectively achieved by HPTLC finger printing technique.¹⁴

Steps involved in HPTLC analysis

a) Sample:

(i) Herbal extract Mixture (HeM): A mixture prepared by combining the individual extracts of all the 11 crude drugs, extracted using authentic crude drugs in the laboratory used in the formulation in the same ratio as given in the formulation.^15,16

(ii) Formulation extract Mixture (FeM): A mixture prepared by combining the extracts obtained from KAPL which are actually used in the manufacturing of the formulation.

(iii) Formulation (F): Madhuniyantrak tablets were crushed and used for analysis.

b) Standard: Curcumin, Emodin, Gallic acid, Ellagic acid and Ascorbic acid.

Preparation of the Sample and standard solution for HPTLC:

Accurately weighed 50 mg of methanol extract of sample (HeM, FeM and F) and standards were weighed in to 10 ml volumetric flask, dissolved in methanol and volume was made up with methanol.

c) Selection of plate and adsorbent: Precoated aluminium plates with silica gel 60 GF₂₅₄ (E.Merck, India) of 20×20 cm and 0.2 mm thickness, were utilized for developing the chromatograph. The plates were pre-washed using methanol and were activated at 60 °C for 5 m prior subjecting to chromatography.

d) Instrument: It consists of CAMAG Linomat V sample applicator, TLC Scanner 3 for detection at 254 and 366 nm and Reprostar 3 for documentation. The Instrumentation was programmed to run through WINCATS 1.4.4 software. Syringe used was 100 μl CAMAG HPTLC syringe.

e) Application of sample: The sample were applied by using 100 μl CAMAG HPTLC syringe. Bandwidth of 2-8 mm was applied on the prewashed TLC plates based on the number of spots applied. Application rate was 50 nl/s.

f) Development: All the plates were developed in CAMAG Twin trough glass chamber with respective optimized solvent system. Saturation time was 20 minutes at room temperature. The developed plates were dried in hot air oven at 60o C for 10 min. then the dried developed TLC plates were scanned at 254 and 366 nm. Based on the absorbance or fluorescence HPTLC fingerprints of extracts were obtained along with the spot of respective standards.¹⁷

Simultaneous identification of all the marker compounds: samples of mixture of extracts, formulation and all the standards were spotted on same TLC plate and developed with optimized solvent system. Developed TLC plate was scanned at 254 and 366 nm and HPTLC fingerprints were obtained.

RESULTS:

Organoleptic characters of extracts used in the formulation:

All the extracts complied with standard requirements with respect to organoleptic properties (Table 1) and proximate analysis (Table 2)

Standardization of extracts as per WHO guidelines:

Extracts of all the crude drugs did not possess any heavy metals, complies with the limit tests for Nickel, Cadmium, Lead and Arsenic. Microbial contents were within the standard limits.

Development of HPTLC technique:

The best solvent system having clear separation of Maximum constituents was selected for each extract and are given in the Table 3.

HPTLC photo documentation of Gymnema sylvestre, Curcuma longa, Azadiracta indica, Momordica charantia, Trigonella foenum graecum, Aegle marmelos, Tribulus terrestris, Emblica officinalis, Terminalia chebula, Terminalia bellerica, Pterocarpus marsupium and Shuddha shilajitare shown in figure 1 to 10. Figure 11 shows 3D Chromatogarm of all the extracts and standards developed by one solvent system.

As per the observations, number of spots and their corresponding rf values for all the extracts, formulation extracts, formulation and standards are given in Table 4.

DISCUSSION:

Ayurveda offers treatment for various diseases through various formulations comprising mostly of crude drugs or plant extracts prepared in a unique way as pre the ancient texts such as Ayurvedic formulary, Baisajya kalpa etc. Churna, Lepa, Lehya, Arista, Asava, Bhasma, Swarasas, Taila, Ghrita are some of the very popular form of ayurvedic formulations. Each type of formulation has its own method of assessment to check its quality, which is again as per the ayurvedic literature. These evaluation patterns include assessing fineness, flotation, stickiness etc. to name a few depending upon type of formulation. But with the influence of modern system of medicine, due to the rapid or quick relief, ease of administration, organoleptic properties, convenience in storage and transportation etc., most of the traditional system of medicines, including Ayurveda have got faded to a great extent. In other hand Allopathy which involves use of synthetically derived chemical substances, antibiotics, organophosphorus, sulpha drugs, steroidal compounds have mild to serious adverse effects, mild to major toxicity to vital organs of the body (Kidney, liver, Nervous system etc.). Treatment for cancer in chemotherapy has disastrous impact on physical and mental status of the patient.

Especially in conditions where long term treatment is required where medicine has become part of daily routine such as epilepsy, thyroid, diabetes etc., patients get loaded with drugs continuously for years together. Due to accumulation of drugs in the body there is a high risk of nephrotoxicity, hepatotoxicity etc.¹⁸

In Diabetic condition, glucose levels are monitored and controlled with most commonly used oral anti-diabetics such as Sulphonyl urea, Biguanides, Thiazolidinediones, Meglitinides etc. which are proved to have serious of side effects.¹⁹ India having history of thousands of years, knowing goodness of Ayurveda, ayurvedic therapies and formulation has lot to offer to alleviate the sufferings of mankind in an economical, safe and effective manner.

Having said all these, there lies a challenge and opportunity for the traditional system of medicines to prove their goodness and make present generation to follow these traditional medicinal systems including Ayurveda. Standardization of medicines used in the traditional systems in terms of modern analytical parameters is much more difficult than that of allopathic medicines due to the presence of hundreds of phytoconstituents. There are few maker compounds present in each plant species. Marker compounds are those constituents which helps in identifying species to species variation and presence of impurities if any. HPTLC is an effective analytical technique which can be utilized to separate these marker compounds and can be identified by comparing R_f values of standards. Each species of plant contains different compounds and hence produces spots at different R_f values in a suitable solvent system. This is very unique and is called as HPTLC fingerprint and the technique used to identify the plant species is called as HPTLC fingerprinting technique.

In the present study, Ayurvedic polyherbal formulation- Madhuniyantrak was standardized by HPTLC fingerprinting technique. “Madhuniyantrak” is an oral tablet manufactured by KAPL, Bengaluru, consists of twelve plant extracts Viz. extract of Gymnema sylvestre, Curcuma longa,Azadiracta indica, Momordica charantia, Trigonella foenum graecum, Aegle marmelos, Tribulus terrestris, Emblica officinalis, Terminalia chebula, Terminalia bellerica, Pterocarpus marsupium and Shuddha shilajit.

Five marker compounds were used for their presence in these extracts. Curcumin with respect to Curcuma longa, emodin with respect to Azadiracta indica, gallic acid with respect to Emblica officinalis, Terminalia chebula, Terminalia bellerica and Aegle marmelos. Apart from these standards, Ascorbic acid and ellagic acid which are usually found in many plant species were also used in order to check their presence in these extracts. The remaining plant extracts were not standardized with respect to their marker compounds due to unavailability of standards.

At first, one solvent system for each of these extracts was optimized to obtain higher resolution in each of these extracts along with formulation and respective marker compounds (excluded in case of standards which were unavailable) (Table No 3). As a further step towards simplification of standardization procedure that is simultaneous identification of all the five marker compounds in the extracts and formulation with one optimized solvent system, mixture of all the extracts prepared in the laboratory, mixture of formulation extracts obtained from KAPL, formulation (Madhuniyantrak) and all the five standards i.e., curcumin, emodin, gallic acid, ellagic acid and ascorbic acid were eluted with optimized solvent system (Sl No 12 in Table No 3). The mobile was optimized based on literatures reviewed with respect to individual standards and trial-error method to elute all the standards in one solvent system. Curcumin and gallic acid were eluted by toluene: ethyl acetate: formic acid (4.5:3.0:0.2, v/v/v) as solvent system²⁰, Gallic acid with ethyl acetate: toluene: acetone (4.5:4:1) and ascorbic acid was eluted with ethanol: glacial acetic acid: toluene (5.5:1:1.5).²¹ Similarly, emodin was eluted with n-hexane-ethyl acetate-formic acid (30:10:0.5)²², Gallic acid and ellagic acid with toluene :ethyl acetate : formic acid : methanol (3:3:0.8:0.4, v/v/v) as mobile phase.²³

From these literatures, information extracted was that there is an overlap of solvents used in these mobile phases. Hence various trial and error was carried out with these solvents and finally an optimized solvent system was developed for simultaneous identification of all these standards and the R_f values of various standards are mentioned in Table No 4, which shows the presence of curcumin, gallic acid, emodin and ellagic acid. Ascorbic acid was found to be absent or less than the limit of detection.

Even though Madhuniyantrak contains twelve drugs, we were successful in identifying five markers in the formulation. Other markers could not be identified due to unavailability. This method is one of its kind for simultaneous identification of curcumin, Ascorbic acid, emodin, Gallic acid and Ellagic acid in a combined herbal formulation which is not been reported so far. This method could be of immense use for evaluation of herbal formulations containing any of these marker compounds individually or in combination in a crude drug, extract or formulate.

SUMMARY AND CONCLUSION:

Experimental results are in support of good quality of the extracts incorporated in ‘Madhuniyantrak’ –herbo-mineral solid formulation indicated for Diabetic condition. We were able to address as aspect of providing validated analytical method to identify and assess quantity of phytomarkers in this polyherbal formulation i.e., HPTLC. This approach could be extended to many other polyherbal formulations also.

CONFLICTS OF INTEREST:

The authors have no conflicts of interest regarding the publication of this article.

ACKNOWLEDGEMENTS:

We Acknowledge Karnataka Antibiotics and Pharmaceuticals Limited, Banglore, for providing samples and drugs required for the present study.