1. INTRODUCTION:

The Indian subcontinent has a large number of medicinal plants used in traditional medical treatments. In India, approximately 70% of the rural population depends on the traditional medical system. Ayurveda is an ancient science of India and helps the human body to keep fit while providing cures from indigenous plants, animal and mineral origin products for various ailments [1]. Ayurveda is a complete and holistic traditional healthcare system of India that contains both preventive and therapeutic aspects [2]. Ayurvedic medicines are usefulness in drug therapy because now a days due to scientific improvement they prove medicinal plants contain more pharmacological active bioactive constituents present in the plants [3]. Phytochemical constituent in the medicinal plants results in the desired healing effect, such as alkaloids, flavonoids, saponins, tannins, alkenyl phenols, terpenoids, phorbol esters and sesquiterpenes lactones. In Ayurveda literature, Berberis aristata has been described as a major plant possess antidiarrheal, antimicrobial, antiprotozoal and antitrachoma activity and used in for several remedies including diarrhea, chronic rheumatism, jaundice, skin diseases, syphilis and urinary disorders. Berberis aristata contains phytoactive constituents such as berberine, oxyberberine, berbamine, aromoline, a protoberberine alkaloid karachine, palmatine, oxycanthine and taxilamine and tannins, sugar, starch [4-7].

Berberine, a benzyl-isoquinoline alkaloid is principal phytoactive constituent possess beneficial pharmacological actions such as in the treatment of wound healing, inflammatory disorders, foe dysentery, skin disease, diarrhea, jaundice, menorrhagia, eye diseases reducing fevers, digestive and respiratory diseases, and microbial pathologies. Additionally, many clinical and experimental studies suggest that berberine has several pharmacological properties, such as metabolic, immunomodulatory, neurological, antioxidative, cardioprotective, hepatoprotective, and renoprotective effects. The genus Berberis represents the around 12 genera and 600 species worldwide and about 77 species have been reported from India [8]. Berberis aristata belongs to berberidaceae family it is one of the most important species due to its extensive medicinal properties and its occurrence has reported from sub-tropical areas (1800-3000 m ASL) of the mountain state of Uttarakhand and Himachal Pradesh in India [9]. The present study has been designed to develop a quality control protocol for the coded Ayurvedic drug by analyzing the reference standard of bioactive compound present in the formulation and respective ingredient. The qualitative and quantitative estimation of major constituent berberine chloride from Berberis aristata stem and in Coded Ayurvedic trial preparation were done by using HPTLC and HPLC. This coded Ayurvedic drug contains twelve ingredients, out of which one ingredient is Berberis aristata DC stem powder. The Chemical structure of berberine chloride has been presented in Figure 1.

Figure 1. Chemical structure of berberine chloride

2. MATERIALS AND METHODS:

2.1 Collection of plant material and Chemicals

Berberis aristata DC. stem and Coded Ayurvedic trial preparation and its ingredients were received from Central Council for Research in Ayurvedic Sciences (CCRAS), New Delhi. Silica gel plate 60 F254 TLC plate of 0.2 mm thickness, chemicals and solvents of analytical grade or HPLC grade were procured from Merck India. Berberine chloride marker compound was purchased from Natural Remedies, Bengaluru, India.

2.2 HPTLC fingerprint profile and analysis

HPTLC analysis was performed on a CAMAG HPTLC system (Muttenz, Switzerland) equipped with an Automatic TLC sampler IV, twin trough development chamber, TLC Scanner 3 linked with WINCATS software version 1.4.4.

Berberis aristata DC stem extract 2ml, Coded Ayurvedic trial preparation drug 10ml and Standard Berberine chloride 2ml solutions respectively were applied on a precoated silica gel 60 F₂₅₄ TLC plate (E. Merck) of 0.2 mm thickness by using Automatic TLC sample applicator (ATS-4). [10-14].

2.3 HPLC instrumentation and chromatographic conditions

HPLC analysis was performed on an Agilent 1200 series equipped with a quaternary pump, manual sample injector, VWD detector and using Chemstation software. All samples and standards were filtered through 0.22 μm membrane filters. Separation was achieved on C18 Eclipse, XDB, 4.6mm x 150mm, 5μm particle size Agilent Column. Different proportions of mobile phase were tried using water, acetonitrile, methanol and phosphate buffer and it also tried isocratic and Gradient elution method. In gradient elution acetonitrile: Potassium hydrogen phosphate Buffer used as mobile phase. Flow rate 1 ml/min at room temperature, 10μl of the test sample and standard was injected into the HPLC system. The detection of analytes at 346 nm was carried out by using VWD. The standard and sample solutions were run in different solvent in various proportions of mobile phase were tried using water, acetonitrile, methanol and Phosphate Buffer and it also tried isocratic and Gradient elution method. Gradient elution method was used for the analysis of Berberine chloride. Flow rate 1 ml/min at VWD 346 nm detection as given a good resolution, sharp and symmetric peak at retention time 3.613 min for standard and nearly same Rt was observed in both samples. The amount of Berberine chloride present in the extracts of Coded Ayurvedic trial preparation and Berberis aristata stem was analyzed by comparing with the standard curve. The calibration curve was drawn by making 0.22mg/ml Berberine chloride stock solution, which was appropriately further diluted to four different concentrations as 0.11, 0.0825, 0.055, 0.0275 mg/ml of working concentrations. Each of the standard solution was run through the HPLC and recorded the respective peak areas [13-18].

3. RESULTS AND DISCUSSION:

Berberis aristata DC stem powder contains Berberine chloride, is one of the major ingredients in Coded Ayurvedic trial preparation . The present study has analyzed the identification and quantification of Berberine chloride present in the Berberis aristata DC stem extract and in Coded Ayurvedic trial preparation and samples extract.

3.1 HPTLC mediated qualitative analysis of berberine chloride in Coded Ayurvedic trial preparation and Berberis aristata DC stem

HPTLC chromatogram gives an understandable picture of standard biomarker whether it is present in Coded Ayurvedic trial preparation and their ingredients. To achieve better separation, optimizations of different mobile phase compositions were employed. Three tracks of test solutions and standards were applied on TLC plate by using an Automatic TLC sample applicator (ATS-4). Chloroform: Methanol: Formic acid (9:1:0.1) solvent system used to develop the TLC plate till the solvent rises to a distance of 8 cm. The developed plate was dried and observed through CAMAG TLC visualizer under UV at 254 and 366 nm, and photos were documented. Berberine chloride band visible in both standard and test tracks at UV 254 nm and 366 nm after photo documentation the plate was scanned using CAMAG TLC Scanner with WINCATS software at a wavelength of UV 254 and 366 nm using deuterium lamps. R_f values were calculated, and photo documentation was recorded in Figure 2 and Table 1.

Once scanning has been completed, the plate was derivatized with vanillin sulphuric acid reagent and heated in a hot air oven at 105°C until the color of the spots appeared and the photo was documented under white light after that the plate was scanned at 540 nm using a tungsten lamp. The HPTLC study evaluated that, A band in 254 nm (Green, R_f 0.05), 366 nm (Fluorescent Green, R_f0.05) and 540 nm (Yellow, R_f0.05) corresponding to Berberine chloride is visible in standard and test solution tracks of Berberis aristata DC stem extract and Coded Ayurvedic trial preparation extract. Details of HPTLC fingerprint profiling are given in Figure 3 [19-23].

At UV 254 nm	At UV 366nm	Derivatized with vanillin sulphuric acid

1 2 3	1 2 3	1 2 3
Track 1-Berberis aristata stem extract; Track 2- Berberine chloride; Track 3- Coded Ayurvedic trial preparation extract

Figure 2. HPTLC fingerprint Profiling of Berberis aristata stem, Coded Ayurvedic trial preparation and Berberine chloride reference standard

Table 1. R_f values of Extracts of Berberis aristata DC stem, Coded Ayurvedic trial preparation and Berberine chloride reference standard

Wave length	Track 1Berberis aristata		Track 2 Berberine chloride			Track 3 Coded Ayurvedic trial preparation
At UV 254nm	R_f	Color	Peak	R_f	Color	Peak	R_f	Color
	0.03	Green	-	-	-	1	0.03	Green
	0.05	Green	1	0.05	Green	2	0.05	Green
	-	-	-	-	-	3	0.10	Green
	-	-	-	-	-	4	0.20	Green
	-	-	-	-	-	5	0.28	Green
	-	-	-	-	-	6	0.31	Green
	-	-	-	-	-	7	0.40	Green
	-	-	-	-	-	8	0.49	Green
	-	-	-	-	-	9	0.55	Green
	-	-	-	-	-	10	0.64	Green
	-	-	-	-	-	11	0.83	Green
	-	-	-	-	-	12	0.87	Green
	-	-	-	-	-	13	0.92	Green
At UV 366nm	0.03	Grey	-	-	-	1	0.03	Grey
	0.05	Fluorescent Green	2	0.05	Fluorescent Green	2	0.05	Fluorescent Green
	0.75	Blue	-	-	-	3	0.15	Blue
	0.87	Blue	-	-	-	4	0.19	Brown
	-	-	-	-	-	5	0.24	Blue
	-	-	-	-	-	6	0.29	Brown
	-	-	-	-	-	7	0.32	Blue
	-	-	-	-	-	8	0.35	Brown
	-	-	-	-	-	9	0.43	Pale brown
	-	-	-	-	-	10	0.50	Red
	-	-	-	-	-	11	0.56	Brown
	-	-	-	-	-	12	0.61	Blue
	-	-	-	-	-	13	0.65	Yellow
	-	-	-	-	-	14	0.70	Brown
	-	-	-	-	-	15	0.74	Pink
	-	-	-	-	-	16	0.78	Brown
	-	-	-	-	-	17	0.81	Blue
	-	-	-	-	-	18	0.83	Yellow
	-	-	-	-	-	19	0.89	Red

Track 1: Berberis aristata stem extract

Track 2: Berberine chloride

Track 3: Coded Ayurvedic trial preparation extract

Figure 3. HPTLC fingerprint Profile of Berberis aristata stem extract, Coded Ayurvedic trial preparation extract and Berberine chloride reference standard at UV 254 nm.

3.2 HPLC mediated quantitative estimation of berberine chloride in Coded Ayurvedic trial preparation and Berberis aristata DC stem extract

Calibration curve was established for peak area Vs concentration of Berberine chloride applied is shown in Figure 4.

The mobile phase combination of acetonitrile and phosphate buffer gradient elution given in the following proportions:

Time	Acetonitrile	Buffer
1	40	60
2	60	40
5	100	0
8	20	80
10	40	60

Figure 4. HPLC Chromatogram of Berberine chloride Standard and Calibration curve

The quantification of bioactive marker compound Berberine chloride in Coded Ayurvedic trial preparation and Berberis aristata stem extracts were performed by using HPLC method. All the samples showed characteristic peaks of Berberine chloride at the same retention time as that of standard Berberine chloride. 10 μl test solutions of Coded Ayurvedic trial preparation and Berberis aristata stem extracts were injected in HPLC system. All determinations were carried out at least three times (n=3) for each extract. Chromatographic peaks of the test solutions of both Extracts were confirmed by comparing their Rt of the reference standard Berberine chloride. Berberine chloride was detected in both extracts. Recorded the chromatogram and calculate the peak area of the test solutions matching to that Berberine chloride as described from the above calibration curve. Results of HPLC showed that standard Berberine chloride is present in Coded Ayurvedic trial preparation and Berberis aristata stem extracts [24-27].

Coded Ayurvedic trial preparation

Berberis aristata DC

Berberine chloride Standard

Figure 5. HPLC Chromatogram of Coded Ayurvedic trial preparation, Berberis aristata DC and Berberine chloride biomarker compound.

The amount of bioactive constituent Berberine chloride present in the Coded Ayurvedic trial preparation and Berberis aristata stem extracts 0.1261% and 5.3935% respectively given in the Figure 5 and Table 3.

4. CONCLUSION

At present, the world is looking forward to embrace herbal/natural products to be safe from untoward side effects of modern medicine allopathy system. In this instance, the use of Ayurvedic drugs becomes more popular because of the scientific improvement to prove medicinal plants contain more pharmacological bioactive constituents present in the plants. In order to get a better or synergistic therapeutic effectiveness, this herbal medicinal formulation may be combined. HPTLC fingerprinting profiling revealed the presence of berberine chloride present in the Coded Ayurvedic trial preparation and in Berberis aristata stem. The R_f value of berberine chloride reference standard 0.05 (Green and Fluorescent Green) was visible in test solution of Berberis aristata stem as well as in Coded Ayurvedic trial preparation extract and found comparable under UV 254 nm and 366 nm, respectively.

The use of markers ensures that the percentages of bioactive components in the herbal mixture are present in reproducible levels in raw materials and in the final dosage form of formulation. HPLC chromatogram concluded that all the samples showed characteristic peaks of berberine chloride at the same retention time as that of standard berberine chloride. The HPLC chromatogram of Berberis aristata DC stem extract and coded Ayurvedic drug corresponding to standard Berberine chloride, showed at a retention time of 3.613 min, at 346 nm wavelengths. The quantitative evaluation revealed the presence of berberine chloride in the coded Ayurvedic drug and Berberis aristata DC stem extract was 0.1261% and 5.3936%, respectively.

5. ACKNOWLEDGEMENTS:

The authors are very grateful to the Director-General, CCRAS, Ministry of AYUSH, New Delhi, for providing encouragement and facilities for carrying out this work.

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Received on 09.10.2021 Modified on 20.11.2021

Research J. Pharm. and Tech 2021; 14(12): 6768-6773.

DOI: 10.52711/0974-360X.2021.01168

S. No	Sample Name	Weight of Extract used for HPLC	Amount of Berberine chloride present in samples	Percentage of Berberine chloride present in sample
S. No	Sample Name	mg/10mL	mg/ml	Result	Mean
1.	*Berberis aristata* Stem	4.35	0.2321	6.2187	5.3936
		4.77	0.199	4.8624
		6.5	0.2844	5.0996
2.	Coded Ayurvedic trial preparation	128	0.1774	0.1740	0.1261
		164	0.214	0.1639
		61	0.0191	0.0393