INTRODUCTION:

Kleeb Bua Daeng formula (KBD) is a Thai traditional herbal formula produced by the Chao Phya Abhaibhubejhr Hospital, Prachinburi Province, Thailand. The main components of this remedy are equal parts Piper nigrum L., Nelumbo nucifera Gaertn. and Centella asiatica L. This formula is used for insomnia symptom and to support cognition and memory. P. nigrum, or black pepper, is a plant in the family Piperaceae that is used as a spice and in traditional medicines. The pharmacological activities of P. nigrum fruit include anticancer, antidepressant, anti-inflammatory, analgesic, antioxidant, hypoglycemic and antimicrobial activities^1-8. This fruit contains many bioactive compounds such as polyphenols, piperine and essential oils^1,9.

The major constituent of P. nigrum is piperine, which shows pharmacological activities suitable for the treatment of depression, Parkinson’s disease and Alzheimer’s disease. N. nucifera is an aquatic plant in the family Nelumbonaceae. This plant has been reported to have anti-inflammatory, antioxidant, neuroprotective, anticancer, and antibacterial activities^10-15. The flowers are used for the treatment of diarrhea, cholera, fever, hyperdipsia, hepatopathy and gastric ulcers^16-18. The bioactive compounds found in the petal part of the flower from this plant are flavonoids (quercetin, rutin, and kaempferol) and anthocyanins (cyanidin-3-O-glucoside)^19-23. C. asiatica, known as Kotu Gola, is medicinal plant in the family Apiaceae that is generally used to promote wound healing and has anti-inflammatory, antioxidant, immunomodulatory, antimicrobial, and neuroprotective properties^24-30. The aerial part of this plant contains active constituents such as asiaticoside, polyphenols, flavonoids, and carotenoids^31-32.

Many previous studies have reported on the determination of active compounds in each of these plants. The most common and useful method for the analysis of these compounds is chromatography, especially high-performance liquid chromatography (HPLC). HPLC is popular as it is an effective instrument for quantitative and qualitative analyses especially in natural and herbal product^33-36. HPLC is an analytical technique suitable for the separation and determination of organic and inorganic solutes in foods, natural products, and pharmaceutical, industrial, biological, and environmental samples. Modern HPLC methods use a non-polar solid phase, like C18, with a polar liquid phase, which is generally a mixture of water and other solvents.

HPLC methods for the quantification of piperine in extracts or powder of P. nigrum fruit^3,4,37,38, quercetin and kaempferol in N. nucifera^16-18,23,39, and asiaticoside and madecassoside in C. asiatica^40-44 have previously been reported. Almost of these HPLC methods used a C18 reverse phase column for the stationary phase and a mobile phase containing an aqueous mixture of an organic solvent with various wavelengths of detection. However, these determinations were carried out separately, analyzing samples taken from one plant at a time⁴⁵. There is no previous report or study about the simultaneous analysis of these active compounds in a mixture of these three herbs, as in Kleeb Bua Daeng formula. Thus, this study aimed to modify these existing HPLC methods to develop a new HPLC method for the simultaneous determination of 5 active compounds in Kleeb Bua Daeng formula so that it can be used for the quality control of this formula. The active constituents that were analyzed were piperine, quercetin, kaempferol, asiaticoside and madecassoside. These active compounds were used as biomarkers for the quality control of Kleeb Bua Daeng formula. The developed HPLC method was validated to ensure the accurate and reliability of the method for the analysis of Kleeb Bua Daeng formula.

MATERIALS AND METHODS:

Materials:

Thai traditional herbal formula used in this study is the KBD Formula, contains 3 herbs: Centella asiatica L. (voucher specimen ABH17), Nelumbo nucifera Gaertn. (voucher specimen ABH15) and Piper nigrum L. (voucher specimen ABH18) and their voucher specimens were deposited at Chao Phya Abhaibhubejhr Hospital Foundation under the Royal Patronage of H.R.H. Princess Bejraratanarajsuda, Prachinburi Province, Thailand. Standards of quercetin, piperine, asiaticoside, madecassoside, kaempferol, tert-Butylhydroquinone (TBHQ), and 3-tert-Butyl-4-hydroxyanisole (BHA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, methanol, and ethanol were purchased from VWR Chemicals BDH (Leicester, England).

Instrumentation:

A gradient high-performance liquid chromatography with diode array detector (Agilent technologies, California, USA) was used for the analysis.

HPLC condition:

The separation was carried out on a reversed-phase HPLC system using a Hypersil ODS C18 column (4.0 mm × 250mm, 5µm, Agilent Technologies Inc., Santa Clara, CA, USA). The sample injection volume was 20 µl, and the column was set at ambient temperature. The detection wavelengths were set at 206nm for madecassoside and asiaticoside, 254nm for piperine, and at 370nm for kaempferol and quercetin. The mobile phase consisted of 0.05% phosphoric acid (A) and acetonitrile (B) at a flow rate of 1ml/min with gradient elution as follows: 0-15min, 80% A; 15-30min, 65% A; 30-35min, 35% A; 35-45min, 20% A; 45-60min, 80% A. Total run time was 60 min.

Preparation of standard solutions:

Madecassoside, asiaticoside, piperine, kaempferol and quercetin were weighed and dissolved with methanol to make stock standard solutions. Working standard solutions were prepared from the stock solutions by dilution with the mobile phase. The concentration of standard solutions ranged from 5 to 30µg/ml for madecassoside, asiaticoside, piperine, kaempferol and from 1 to 6µg/ml for quercetin. All standard solutions were filtered through a 0.45 µm nylon syringe filter membrane before chromatographic analysis. All solutions were stored at 4°C.

Sample preparation:

a) Preparation of sample solutions for the analysis of quercetin and kaempferol:

The hydrolysis of KBD formula extract was modified from the method of Nuutila in 2002 and Panya in 2018^46-48. Fifty milligrams of KBD formula extracts were hydrolysed in 5ml of 1.2 M HCl in 50% aqueous methanol, 5mg of TBHQ and 5 mg of BHA were added. After that, refluxed at 80°C for 2 hours. The extract was allowed to cool the volume was adjusted to 10ml with methanol, and the mixture was sonicated for 30minutes. After hydrolysis, the extracts were filtered through a 0.45µm nylon syringe filter membrane, mixed with mobile phase in the same dilution and 20µl samples were injected into the HPLC system.

b) Preparation of sample solutions for the analysis of madecassoside, asiaticoside and piperine:

Ten milligrams of KBD formula extracts were dissolved in 1ml of methanol and the volume was adjusted to 10 ml with mobile phase. After that, the extract solutions were sonicated for 15 minutes and filtered through a 0.45µm nylon syringe filter membrane and 20µl samples were injected into the HPLC system.

Method validation:

The analytical method was validated according to the ICH guidelines (International Conference on Harmonization)⁴⁹. The validation parameters included specificity and sensitivity, linearity, range, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and robustness.

a) Specificity:

The specificity of the method was evaluated by injecting 20µl solutions of standard, sample, and blank into the chromatographic system.

b) Linearity:

The linearity was validated by preparing standard solutions at five concentrations. Working standards were prepared in the range of 5–30µg/ml for madecassoside, asiaticoside, kaempferol, piperine and 1-6µg/ml for quercetin. Twenty microliters of each concentration were injected into the HPLC system. The peak area against concentration was subjected to regression analysis to calculate the calibration equations and coefficient of determination.

c) Precision:

The precision of the method was determined by repeatability (within-day) and intermediate precision (between-day). The within-day precision is expressed as the percent relative standard deviation (%RSD) during the same day. The between-day precision was studied by comparing the assays on five different days and the standard deviation (SD) and %RSD were calculated.

d) Accuracy:

The accuracy was evaluated by successive analysis (n = 3) of three different concentrations of madecassoside, asiaticoside, piperine, kaempferol (5, 15 and 30µg/ml) and quercetin (1, 3 and 6µg/ml) using the developed method. It was obtained by calculating the percent recovery (%recovery). The mean recovery should be within 90–110% to be acceptable.

e) Limit of detection (LOD) and limit of quantification (LOQ):

Limit of detection (LOD) is the lowest concentration in a sample that can be detected. LOD was established at a signal-to-noise ratio of 1:3. Limit of quantitation (LOQ) is the lowest concentration that can be determined with acceptable precision and accuracy. LOQ was established at a signal-to-noise ratio of 1:10.

f) Robustness:

The robustness of the method was evaluated by carrying out the analysis using different flow rates (±0.1ml/min) and keeping the other chromatographic conditions constant.

RESULTS:

Accuracy:

The results of accuracy are expressed as percent recovery (% recovery) and are presented in Table 1. The mean percentage recoveries of madecassoside, asiaticoside, piperine, kaempferol and quercetin were found to be in the range of 99.09 to 100.66%, 98.95 to 101.85%, 98.14 to 101.18%, 99.22 to 102.07% and 100.63 to 102.41%, respectively. The results of percentage recovery were within accepted limits of 90.0% to 110.0%.

Table 1. Percentage recovery of madecassoside, asiaticoside, piperine, kaempferol and quercetin

Compounds	Concen tration (µg/ml)	Concentration founded (µg/ml)	%Recovery (n=3)
Madecassoside	5	5.03 ± 0.62	100.66 ± 1.24
	15	14.86 ± 0.15	99.09 ± 0.10
	30	30.10 ± 0.11	100.34 ± 0.37
Asiaticoside	5	5.09 ± 0.06	101.85 ± 1.20
	15	14.78 ± 0.13	98.55 ± 0.88
	30	30.16 ± 0.06	100.52 ± 0.20
Piperine	5	5.06 ± 0.02	101.18 ± 0.30
	15	14.72 ± 0.02	98.14 ± 0.13
	30	29.78 ± 0.01	99.26 ± 0.02
Kaempferol	5	5.07 ± 0.01	101.42 ± 0.11
	15	15.31 ± 0.01	102.07 ± 0.30
	30	29.77 ± 0.01	99.22 ± 0.02
Quercetin	1	1.02 ± 0.01	102.41 ± 0.70
	3	3.02 ± 0.09	100.63 ± 0.30
	6	6.04 ± 0.01	100.64 ± 0.05

Precision:

The within-day and between-day precision of the method is expressed as the percent relative standard deviation (% RSD) value. Precision was indicated at three concentration levels in the precision studies. Within-day precision was determined by injection of standard solutions on the same day. Between-day precision was checked by repeating the studies on five different days. The %RSD values for within-day and between-day assays of all compounds indicated good precision (<2 %). The acceptable precision value was not more than 2.0% for the RSD. The results are shown in Table 2.

Table 2. Within-day and between-day precision data of madecassoside, asiaticoside, piperine, kaempferol and quercetin

Compounds	Concentration (µg/ml)	Within-day precision (n=3)		Between-day precision (n=5)
Compounds	Concentration (µg/ml)	*Area of peak (mAUS)**	%RSD	*Area of peak (mAUS)**	%RSD
Madecassoside	5	21.45 ± 0.42	1.94	21.14 ± 0.34	1.61
	15	87.35 ± 1.00	1.15	88.07 ± 1.02	1.15
	30	189.49 ± 0.75	0.40	191.94 ± 0.44	0.23
Asiaticoside	5	28.01 ± 0.39	1.40	27.94 ± 0.26	0.92
	15	91.46 ± 0.86	1.43	90.90 ± 0.60	0.66
	30	192.13 ± 0.39	0.20	193.18 ± 0.97	0.50
Piperine	5	234.13 ± 0.77	0.33	235.12 ± 1.15	0.49
	15	727.33 ± 1.01	0.14	728.92 ± 1.17	0.16
	30	1495.87 ± 0.23	0.02	1497.32 ± 0.89	0.06
Kaempferol	5	584.49 ± 0.70	0.12	585.46 ± 1.11	0.19
	15	2545.76 ± 0.58	0.03	1927.08 ± 0.80	0.04
	30	3819.67 ± 0.66	0.02	3821.55 ± 1.36	0.04
Quercetin	1	89.35 ± 0.67	0.75	89.91 ± 0.77	0.85
	3	278.89 ± 0.86	0.31	281.75 ± 1.07	0.38
	6	565.80 ± 0.27	0.05	566.98 ± 0.59	0.10

Table 3. Regression equation, correlation coefficient linearity ranges, limits of detection (LOD) and limits of quantification (LOQ) of calibration curves for the five compounds

Compounds	Regression equation	Coefficient of determination (R²)	Linearity range (µg/ml)	LOD (µg/ml) (n=3)	LOQ (µg/ml) (n=3)
Madecassoside	y = 6.7031x - 12.284	0.9997	5-30	2.90 ± 0.46	7.00 ± 0.75
Asiaticoside	y = 6.5481x - 5.3367	0.9996	5-30	4.23 ± 0.59	9.77 ± 0.99
Piperine	y = 51.042x - 24.099	0.9990	5-30	4.23 ± 0.66	8.80 ± 1.30
Kaempferol	y = 131.01x - 79.883	0.9992	5-30	5.80 ± 0.35	13.63 ± 1.85
Quercetin	y = 95.012x - 7.948	0.9997	1-6	5.85 ± 1.06	12.10 ± 0.82

Table 4. Results of the robustness study from the variation of flow rate

Compounds/Flow rate	0.9 mL/min		1.0 mL/min		1.1mL/min
Compounds/Flow rate	value	%RSD	value	%RSD	value	%RSD
Madecassoside
Retention time	12.37	0.43	11.64	0.04	10.89	0.79
Peak area	91.05	0.24	88.69	1.18	82.99	0.84
%Recovery	102.77	0.21	100.43	1.04	94.75	0.74
Asiaticoside
Retention time	14.74	0.49	13.97	0.03	13.21	0.77
Peak area	99.27	0.75	91.56	0.61	89.43	0.62
%Recovery	106.50	0.72	98.65	0.57	96.49	0.59
Quercetin
Retention time	16.04	0.29	15.73	0.02	14.55	0.20
Peak area	291.09	0.21	278.89	0.29	248.56	0.17
%Recovery	104.91	0.20	100.63	0.29	89.99	0.16
Kaempferol
Retention time	20.66	0.31	20.37	0.03	19.17	0.06
Peak area	2268.64	0.05	1928.05	0.03	1849.56	0.04
%Recovery	119.51	0.04	102.18	0.03	98.18	0.05
Piperine
Retention time	29.16	0.14	28.45	0.08	27.51	0.01
Peak area	788.60	0.72	728.78	0.18	661.25	0.10
%Recovery	106.15	0.70	98.33	0.17	89.51	0.10

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The results showed the LOD for madecassoside, asiaticoside, piperine, kaempferol and quercetin were 2.90, 4.23, 4.23, 5.80 and 5.85µg/ml, respectively and the LOQ of these compounds were 7.00, 9.77, 8.80, 13.63 and 12.10µg/ml, respectively (Table 3).

Robustness:

The robustness of the method was evaluated by changing the flow rate. Acceptable robustness was not more than 2.0% for the RSD. The results showed that varying the flow rate had no appreciable effects on the chromatographic parameters (Table 4).

Determination of madecassoside, asiaticoside, quercetin, kaempferol and piperine in Kleeb Bua Daeng extract:

Chromatograms of madecassoside, asiaticoside, piperine, kaempferol and quercetin detected under the developed HPLC conditions are shown in Figure 2. It can be seen from the figure that good separation was achieved within 30 min. The retention times for madecassoside, asiaticoside, quercetin, kaempferol and piperine were 11, 13, 15, 19 and 28 min, respectively.

Figure 2. HPLC chromatogram of standard madecassoside and asiaticoside solution (conc.30 µg/ml) at wavelength 206 nm (A), piperine solution (conc.30 µg/ml) at wavelength 254 nm (B), quercetin (conc. 6 µg/ml) and kaempferol solution (conc.30 µg/ml) at wavelength 370 nm (C)

The developed and validated analytical HPLC method was applied for the analysis of madecassoside, asiaticoside, piperine, kaempferol and quercetin simultaneously in KBD formula extract. The peak areas of triplicate samples were analyzed by a regression equation obtained from the calibration plot to get the madecassoside, asiaticoside, piperine, kaempferol and quercetin contents in the samples. The KBD formula extracts were found to contain 45.25mg/g extract of madecassoside, 182.06mg/g extract of asiaticoside, 1.09 mg/g extract of quercetin, 1.96mg/g extract of kaempferol and 123.98mg/g extract of piperine (Table 5). The peaks of madecassoside, asiaticoside, piperine, kaempferol and quercetin in the sample were identified by comparing the retention time with that of standards and confirmed by comparing with their respective UV spectra (Figure 3).

Figure 3. HPLC chromatogram of KBD formula extract at wavelength 206 nm (A), 254 nm (B) and 370 nm (C)

Table 5. Quantitative analysis of five active compounds in KBD formula extract by the developed HPLC method

Compounds	Content (mg/g extract) (n=3)
Madecassoside	45.25 ± 0.83
Asiaticoside	182.06 ± 3.11
Quercetin	1.09 ± 0.06
Kaempferol	1.96 ± 0.01
Piperine	123.98 ± 0.15

DISCUSSION:

HPLC chromatograms from all standard solutions showed good separation of peaks, with resolution values of more than 2. The within-day and between-day percentage relative standard deviations (% RSD) for five replicate injections were all less than 2%. The calibration curve obtained from plotting the peak areas at each concentration showed good linearity (correlation coefficient r² > 0.99). Accuracy, as determined by the percentage recovery of spiked standards, was always between 90% and 110%. The remaining validation data obtained for this HPLC method (limit of quantitation; LOQ and limit of detection; LOD and robustness) are presented. From all validation parameters, it was found that this analytical method was reliable and could be used for the analysis of these five compounds. This HPLC method was then used to determine the amount of each of the five active compounds in ethanol extracts of Kleeb Bua Daeng formula. Notably, these chemical marker constituents of Kleeb Bua Deng formula are needed in the quality control process to standardize the herbal extract and the formula. Therefore, developing an appropriate and efficient method to determine the chemical marker quality control of the formula was an important and critical issue. The major compounds found in this formula, and selected in this study, were determined by this developed HPLC method. This study focused on the content of piperine, quercetin, kaempferol, asiaticoside and madecassoside in KBD formula due to the neuroprotective effects of these compounds. Piperine, a major constituent isolated from P. nigrum, has been reported by Yang and co-workers⁵⁰ to improve cognition and exert a protective effect on dopaminergic neurons via antioxidant, anti-apoptotic, and anti-inflammatory mechanisms in an MPTP-induced mouse model of Parkinson’s disease. Another study indicated that methanolic extract of P. nigrum fruits had anxiolytic and antidepressant effects and might confer neuroprotective effects due to the alleviation of oxidative stress induced by Aβ (1–42) injection in the rat amygdala³. Other active constituents in KBD formula are quercetin and kaempferol from N. nucifera. A previous study revealed that quercetin showed significant antidepressant and cardioprotective effects⁵¹. Moreover, the antidepressant-like effects of quercetin were independent of the hypothalamic-pituitary-adrenal axis and quercetin was found to decrease extracellular β-amyloidosis, tauopathy, astrogliosis and microgliosis in the hippocampus and the amygdala^52-53. In addition, quercetin has been shown to significantly attenuate rotenone‐induced behavioural impairment, augment autophagy, ameliorate ER stress‐induced apoptosis and attenuate oxidative stress⁵⁴. Kaempferol also exerts antidepressive effects, which might be mediated, at least in part, by enhancing antioxidant capacity and by anti-inflammatory effects via up-regulation of AKT/β-catenin cascade activity in the prefrontal cortex of CSDS mice⁵⁵. Kaempferol also performed as a neuroprotective agent in a rotenone-induced rat PD model and in SH-S5Y5 cells by preventing the loss of tyrosine hydroxylase expression⁵⁶. Asiaticoside and madecassoside, the major compounds in C. asiatica, might also play a role as antidepressant and anti‑inflammatory agents, which was shown via their selective inhibition of MAO-A over MAO-B and by attenuating neurotoxicity^57-60. Taken together, these active compounds are appropriate biomarkers of Kleeb Bua Daeng formula. Quantification of these active compounds in KBD formula by the developed and validated HPLC method could be applied to confirm the consistency and potential activity of this herbal product across different batches or lots during manufacturing. This HPLC method has an advantage over the previous HPLC method by Maneenet and coworkers⁴⁵ as it is able to simultaneously analyse five key active compounds in the KBD formula. Thus, the method developed in this study is more widely applicable and saves analytical time.

CONCLUSION:

A reliable and accurate HPLC method has been developed and validated to determine five active compounds including piperine, kaempferol, quercetin, asiaticoside and madecassoside in KBD formula. It proved suitable to be applied for routine quality control analysis of KBD formula. The analytical method conditions provide good resolution for these five active compounds. The method was validated in accordance with ICH guidelines and passed all important parameters including robustness. The outcome of these findings is likely to be useful as a quality control pattern for the standardization of this formula.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Khon Kaen University (KKU RP: F64010098) for research funding and Dr. Glenn Borlace for English language assistance through the Khon Kaen University publication clinic.

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Received on 23.07.2021 Modified on 27.11.2021

Research J. Pharm. and Tech. 2022; 15(8):3618-3626.

DOI: 10.52711/0974-360X.2022.00606