1. INTRODUCTION:

From the series of isoquinoline alkaloids, Berberine (BER) possesses an extensive history of medicinal use in ayurvedic, Chinese, and Unani medicinal systems found majorly in the stem bark, rhizomes, and roots of Berberis aristata, Berberis aquifolium (Oregon grape) and Berberis vulgaris (barberry)^1-3. Pharmacologically, BER has potential to inhibiting acetylcholinesterase (Ach ase), lowering glucose and cholesterol, reducing mortality rate in patients with chronic congestive heart failure (CHF)³.

Moreover, BER and B. aristata extract is known to be good anti–bacterial, anti-protozoal, anti-inflammatory, anti-hepatotoxic, anticancer, and wound healing agents, etc. Therapeutically, BER is also used in the treatment and cure of coronary artery disease, diabetes, hyperlipemia, ischemic stroke, etc.⁴ Curcumin (CUR), a yellow-colored polyphenol derived from the rhizome of Curcuma longa, belonging to the ginger family i.e., zingiberaceae generally named as termeric.

Turmeric has been used for medicinal purposes in ayurveda for over 6000 years. It contains curcuminoids (4-6%), fixed oil (2-3%), essential oil (2-4%), and a mixture of volatile oils such as atlantone, turmerone, and zingiberone. Curcumin is the primary curcuminoid found in turmeric and has a distinct earthy, slightly bitter, warm peppery flavor and a mustardy odor. Curcuma longa also contains a variety of active phytochemicals like bisdemethoxycurcumin, demethoxycurcumin, tetrahydro curcumin, zingiberene, eugenol, curcumenol, triethyl curcumin, curcumol, turmerin, turmerones, and turmeronols. Curcumin, desmethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are collectively known as curcuminoids. Curcumin has been shown to have various pharmacological benefits such as anti-inflammatory, anticancer, antioxidant, and antimicrobial effects. It also has chemo preventive properties and can suppress tumorigenic activity in several types of cancer. Curcumin is also beneficial in treating other diseases like diabetes, Alzheimer's disease, Parkinson's disease, and arthritis. Curcumin and xanthorrizol are marker compounds in curcuma rhizomes and can be used for standardization of plants, extracts, and polyherbal formulations. Recent studies suggest that a combination of curcumin and berberine or their corresponding plant extracts possess several pharmacological benefits. Additionally, there are several formulations available in the market containing extracts of Berberis aristata and Curcuma longa as active ingredients. Together, these extracts seek to help in maintaining optimal health and to support a healthy internal response. These formulations standardized using their corresponding active markers like BER and CUR. Literature survey of last 10 years revealed that a number of analytical approaches e.g., HPLC¹⁹, HPTLC²⁰, UV-Visible²¹ and Fluorometric²² methods developed for single constituent estimation but there is no reliable RP-HPLC method available for simultaneous valuation of BER and CUR in polyherbal formulation or mixture of extracts. The purpose of this research work was to develop and validate reliable procedure for the simultaneous determination of CUR and BER in their pure, combined extracts and marketed formulations without any prior separation of individual drugs.

2. MATERIALS AND METHODS:

Berberis aristata and Curcuma longa were collected from the herbal garden of Ch. Devi Lal College of Pharmacy in Jagadhri, Haryana and were authenticated in the Pharmacognosy Department of the institute. Herbal markers BER (CAS-633-65-8) and CUR (CAS-458-37-7) were procured from Sigma Aldrich. Ortho-phosphoric acid (H3PO4, 85%), Potassium dihydrogen phosphate (KH2PO4) of analytical-reagent (AR) grade and methanol (CH3OH), acetonitrile (ACN) of HPLC grade were bought from Central Drug House (P) Ltd. in New Delhi. An herbal formulation named ‘Solaray’ which is a dietary supplement containing berberine and curcumin root extracts (300mg/capsule) was purchased from the local market in Yamuna Nagar, Haryana.

2.1 Instruments:

The HPLC used was a Waters system from Milford, USA. It was composed of a 515 solvent delivery module, PDA-2998 detector, Waters 2707 autosampler, injector with a 20µl loop and Agilent HC C-18(2) column (particle size 5µm; 250mm×4.5mm SN-usjab01584). Chromatographic results were measured and interpreted using EMPOWER-2 software. To prepare the HPLC grade water, a water Elix® 03 unit from Millipore, USA was used.

2.2 Extraction procedure for Berberis aristate:

The stem bark of B. aristata was collected, cut into small pieces, dried in the shadow and powdered. Powdered content of B. aristata was treated with petroleum ether (100mL) in a soxhlet extractor unit for 12hrs at 60°C. Further air-dried the marc and extracted with CH₃OH for the same conditions The marc was air-dried and further extracted with methanol in a soxhlet for 12hrs at 60°C. At the end, filtered and concentrated the extract to dryness at <60°C under pressure-controlled conditions by a rota evaporator. Then residue was diluted with CH₃OH and used for quantification of BER content using RP-HPLC²³.

2.3 Extraction procedure:

The process involved collecting dried rhizomes of C. longa and cutting them into small pieces. Then, 500 grams of these small pieces were refluxed with methanol in a reflux condenser for two and a half hours. The resulting extract was cooled to room temperature and quantitatively filtered into a volumetric flask. It was then concentrated at a temperature of less than 60°C under reduced pressure using a rotary evaporator, and finally diluted up to the mark of 100mL with methanol. This extract was then used to quantify the CUR content through RP-HPLC.²⁴

2.4 Sample preparation:

A solution of BER (1000µg/mL) and CUR (1000 µg/mL) in methanol was prepared. To obtain 10µg/mL of BER and CUR, aliquots of the above-mentioned solutions were diluted stepwise with mobile phase, and UV-Vis’s spectra were recorded (Fig. 1). These solutions were then used to simultaneously assess the components using the proposed HPLC procedure. A stock solution of a marketed formulation was also prepared and diluted in a similar manner to the plant extracts and corresponding pure herbal markers.

2.5 Optimization of chromatographic conditions:

Following conditions were studied for optimum separation such as: (i) Various organic solvents used such as methanol, ACN with water (ii) Various buffers were used such as Triethylamine (TEA, 0.01%v/v), Ortho-phosphoric acid (OPA, 10%v/v) and KH₂PO₄(iii) Various molarities of KH₂PO₄were used such as 20mM, 30mM and 50mM (iv) Mobile phase composition ACN: KH₂PO₄ (20mM) varied at 70:30, 65:35 and 60:40 (v) pH of aqueous phase varied by 2.5, 3 and 3.5 keeping the composition of ACN: KH₂PO₄ (20mM) 65:35 and ﬂow rate;1.0mL/min (vi) Variable flow rate (0.8, 1.0, and 1.2mL/min) with mobile phase(ACN: KH₂PO₄) ratio and pH maintained at 65:35 and 3, respectively. Moreover, the effects of different level of all these three factors (Variation in mobile phase ratio, flow rate and pH) were systematically addressed on system suitability parameters e.g., resolution, retention time (Rt), peak area, theoretical plates, separation factor, capacity factor, asymmetry etc.

To optimize and develop the method, all mobile phases were prepared and their pH was adjusted using 10% OPA. Once the pH was adjusted, the mobile phase was filtered through a 0.22µm membrane filter and sonicated for at least 9 minutes before being used for chromatography.

2.6 System suitability parameters:

An integral part of HPLC method are system suitability tests (SST), used to authenticate the resolution and reproducibility of the chromatographic system is suitable for analysis. These tests check and validate complete testing system including the equipment, electronics, column, reagents, analytical operations and samples to be analyzed²⁵.

2.7 Validation of optimized method:

Once the chromatographic conditions have been optimized and developed (as shown in Fig. 2), it is necessary to validate and authenticate the method. This involves verifying that the features of the procedure meet the requirements of the application domain. The developed method should be validated according to the ICH Guidelines for linearity, range, precision, accuracy, sensitivity, limit of detection (LoD), limit of quantification (LoQ), and percentage recovery. This ensures that the method is reliable and suitable for its intended use.²⁵

2.8 Calibration curve (Linearity):

The parameter that establishes a directly proportional relationship between the concentration of an analyte and its response is referred to as linearity. This is represented in a calibration curve. In this study, a calibration plot was created by performing a replicate analysis (n=6) at all concentration levels. The linear relationship was evaluated using the least square method within the Microsoft Excel® program. The peak area ratio of BER and CUR was used in this analysis.

2.8.1 Precision:

Intra-day precision and inter-day precision protocol was determined in terms of percent relative standard deviation (% RSD). The experiments were repeated three different days and three different times in a day for inter-day and intra-day precision respectively and % RSD for both inter-day and intra-day precision were calculated.

2.8.2 Percentage purity:

Twenty capsules were weighed accurately and their average weight was calculated. The content of the capsules was then refluxed in 50mL of methanol for half an hour. The extracted solution was cooled and quantitatively filtered into a 100 mL volumetric flask, which was then filled up to the 100mL mark with methanol. This final extract can be used to determine the percentage purity in HPLC.

2.8.3 Accuracy:

Accuracy reflects the difference and relation between measured/practical value and real/theoretical value. To check the accuracy of the developed method, analytical recovery experiments were performed by following standard addition method using 50%, 100% and 150% of the marker. After three times repetition of each concentration, accuracy was calculated as % R.S.D.

2.8.4 Sensitivity:

As per IUPAC and ISO²⁶, LOD and LOQ were determined according to following equation:

ksB

LOD or LOQ = ––––––

where sB is the standard deviation of the analytical signal, k is a constant (3 for LOD and 10 for LOQ) and S is the slope of the concentration/response graph.

2.8.5 Speciﬁcity: Speciﬁcity protocol express the effect of interferences on results of developed method. The speciﬁcity was determined by evaluating the comparison of the chromatograms got from the samples containing BER and CUR with those measured from blank.

2.8.6 Robustness: A small but systematic change like variation in composition of mobile phase and wavelength range etc. In present study, assessment of BER and CUR were studied by means of change in mobile phase composition, mobile phase volume and deviations in the results were calculated as % RSD.

3. RESULTS AND DISCUSSION:

The proposed method was developed and the optimized chromatographic conditions such as optimum mobile phase was found to be ACN: KH₂PO₄(20mM) in ratio of 65:35 adjusted to pH 3.0 at flow rate of 1 ml/min (Table 1). A good sensitivity and resolution of the method were observed for BER and CUR at 272 nm. Typical chromatogram (Fig. 2) with optimized conditions gives sharp and symmetric peak with Rt of 3.0 and 5.4 min. for BER and CUR respectively. These results show that the compounds eluting rapidly, saving time while running this optimized method.

Fig. 2: Chromatogram of BER and CUR for optimized HPLC method [Optimized Chromatographic Conditions are Stock solution: Methanol, Stationary phase: Agilent HC C-18 column (paricle size 5µm; 250mm×4.5mm), Mobile phase: ACN:20 mM KH₂PO₄ (65:35), Flow rate: 1.0mL /min, pH: 3.0]

3.1 System Suitability Test (SST):

The validation report directed that proposed developed RP-HPLC method is simple, sensitive, specific, accurate, more precise, less time consuming and can be convenient for routine analysis^27-30 and characterization of BER and CUR in bulk drugs and pharmaceutical formulations. Table 2 shows SST for BER and CUR used for validation of proposed RE-HPLCmethod respectively.

Table 2: SST for BER and CUR

SST Parameters	BER	CUR
Area	309335.8	270714.7
Tailing factor (T_f)	1.2	1.8
Capacity factor (k)	0.23	1.1
Separation factor (α)	-	1.79
Resolution (R_s)	-	5.57
Theoretical plates USP	2912	2331

3.2.1 Calibration curve (linearity):

A calibration curve was produced by injecting a range of six different concentrations of BER and CUR solutions. The high coefficient of determination values (r2)^31-34, which were 0.9990 for BER and 0.9990 for CUR (r2 > 0.995), indicated good linearity between their peak areas (y) and standard drug concentrations (x, μg/ml) within the range of 150-4800 ng/ml for BER and 900-28800 ng/ml for CUR, respectively (as shown in Fig. 4). The overlay of peaks is illustrated in Fig. 3.in the range 150- 4800 ng/ml for BER and 900-28800 ng/ml for CUR respectively (Fig. 4) and overlay of peaks are represented in Fig. 3.

Fig. 3: Overlay of BER and CUR peaks by optimized RP-HPLC method

Fig. 4: Linearity for BER and CUR

3.2.2 Precision:

% RSD for intra and inter-day precision were found to be 1.667 and 0.887 for BER and 0.954 and 0.718 for CUR (% RSD.< 2.0) respectively which indicated that the proposed method is highly precise and reproducible (Table 3).

Table 3: Precision report of RP-HPLC method for BER and CUR

Conc. of BER (ng/ml)	Precision(%R.S.D.)^a		Conc. of CUR (ng/ml)	Precision(%R.S.D.)^a
Conc. of BER (ng/ml)	Intraday	Interday	Conc. of CUR (ng/ml)	Intraday	Interday
300	1.275	1.216	1800	0.911	0.961
1200	1.942	0.462	7200	1.201	0.846
4800	1.783	0.923	28800	0.745	0.395
Mean	1.667	0.887	Mean	0.954	0.718

^a Mean and standard deviation for 3 determinations

3.2.3 Percentage purity determination:

The proposed method used for the quantification of BER and CUR in plant extracts and marketed capsules. Satisfactory results were measured in analysis of both herbal markers and were found in good agreement with the label values (Table 4). Moreover, for the validity of the proposed methods, the standard addition method (SAM) used by adding reference BER and CUR to the previously analyzed marketed capsules. The recovery value of each marker was calculated by comparison of the concentration recovered from the spiked mixtures against the pure marker.

Table 4: Percentage purity of BER and CUR in plant extracts and formulation.

Sample

Marker

% w/w Content

Mixture of extract of B. aristata and C. longa

BER

CUR

0.2 ± 0.3

4.0±2.0

Formulation- Solaray

BER

CUR

94.61

95.94

3.2.4 Accuracy:

Table 5 shows that the proposed method produced high recovery rates of 98.15±0.47 % w/w for BER and 98.57 ±0.98 % w/w for CUR respectively.

Table 5: Accuracy results of RP-HPLC method for BER and CUR

Amount added (ng/ml)		% Recovery^a
BER	CUR	BER	CUR
1440	21600	99.26	99.01
1920	28800	96.26	98.15
2400	36000	98.91	98.56
Mean Recovery		98.15	98.57
%R.S.D.		1.76	1.23

^a Mean and standard deviation for 3 determinations

3.2.5 Detection and Quantification limits:

The LOD and LOQ were found to be 50 and 150 ng/ml for BER and 300 and 900 ng/ml for CUR. These data showed highly sensitive and specificity of proposed method.

3.2.6 Robustness and Ruggedness:

Robustness and ruggedness were listed in Table 6 for BER and CUR, which shows that the proposed method was robust.

Table 6: Robustness and Ruggedness parameter for BER and CUR

Parameters	Variations	%RSD for Area (n=6)		%RSD for Retention time (n=6)
Parameters	Variations	BER	CUR	BER	CUR
Mobile phase	+2%	1.04	1.12	0.98	1.75
	-2%	0.82	0.91	0.46	0.84
	Normal	0.887	1.667	0.986	1.72
Flow rate	+10%	1.44	1.98	1.03	0.39
	-10%	1.76	1.22	1.86	0.74
	Normal	0.98	1.41	1.11	1.53
pH	+0.2unit	1.77	0.41	0.91	0.74
	-0.2unit	1.34	0.74	1.20	1.36
	Normal	1.12	1.64	0.99	1.79
Detection wavelength	+0.2unit	1.44	1.75	0.64	0.74
	+0.2unit	1.78	0.84	1.59	1.12
	Normal	1.21	1.82	1.34	1.68
Analyst	Analyst 1	0.46	0.84	1.59	1.98
Analyst	Analyst 2	1.78	0.75	1.44	1.74

4. CONCLUSION:

Analysis of drug present in polyherbal formulation is a quite challenging problem. The newly developed RP-HPLC method for analysis of BER and CUR simultaneously is simple, specific, accurate, precise, rapid, economical and robust; indicates its adequacy for routine pharmaceutical analysis of BER and CUR in plant extracts (Berberis aristata and Curcuma longa) and marketed polyherbal formulations (Table 7).

Table 7: Validation parameters for BER and CUR

Parameters	BER	CUR
Absorption maxima (nm)	272	272
Linearity range (ng/ml)	150- 4800	900-28800
Coefficient of determination(r²)	0.9990	0.9990
Regression equation (Y^a)	Y = 186.4x + 15658	Y = 28.23x + 3900
Slope (m)	186.4	28.23
Intercept (c)	15658	3900
LOD (ng/ml)	50	300
LOQ (ng/ml)	150	900
Accuracy(%w/w)	98.15	98.57
Precision (%RSD) Intra-day Inter-day	1.667 0.887	0.954 0.718

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Received on 07.06.2024 Revised on 12.08.2024

Accepted on 19.09.2024 Published on 22.10.2024

Available online from October 31, 2024

Asian J. Research Chem. 2024; 17(5):264-270.

DOI: 10.52711/0974-4150.2024.00046

Chromatographic parameters	Variation	Peak shape
M. P. composition: Changes in mobile phase composition in different ratio of ACN:KH₂PO₄ with keeping constant with flow rate of 1.0 ml/min fixed.	70:30	Peak merged
	65:45	Sharp peak, poor symmetry
	60:40	Broad peak, R_tshifts
pH variation: Change in pH with mobile phase composition 65:35 of ACN: KH₂PO₄at fixed flow rate (1 ml/min).	3.5	Broad peak, tailing, R_tshifts
	3.0	Sharp peak not tailed
	2.5	Sharp peak, tailing
Flow rate variation: Changes in flow rate with mobile phase composition 65:35 of ACN: KH₂PO₄and pH maintained at pH 3.0	1.2 1.0 0.8	Peak merged Sharp peak, less tailing Broad peak, poor symmetry, tailing