Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 
  • Users Online: 125
  • Home
  • Print this page
  • Email this page


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 16  |  Issue : 3  |  Page : 227-239

Validation of an HPLC method for quantification of anti-inflammatory markers in an ethanolic extract of Sahastara and its anti-inflammatory activity in vitro


1 Faculty of Medicine, Thammasat University, Pathumthani, 12120, Thailand
2 Department of Applied Thai Traditional Medicine, Faculty of Medicine; Center of Excellence in Applied Thai Traditional Medicine Research, Thammasat University, Pathumthani, 12120, Thailand
3 Regional Medical Science Center 12 Songkhla, Department of Medical Sciences, Songkhla, 90100, Thailand
4 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada

Date of Submission06-Dec-2020
Date of Decision11-Jan-2021
Date of Acceptance20-Jan-2021
Date of Web Publication12-May-2021

Correspondence Address:
Arunporn Itharat
Department of Applied Thai Traditional Medicine, Faculty of Medicine; Center of Excellence in Applied Thai Traditional Medicine Research, Thammasat University, Pathumthani, 12120
Thailand
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1735-5362.314821

Rights and Permissions
  Abstract 


Background and purpose: Sahastara (SHT) is a traditional Thai medicine for the treatment of musculoskeletal and joint pain. It consists of 21 plant components. A previous study demonstrated the anti-inflammatory activity of SHT on inhibition of nitric oxide production and prostaglandin E2 (PGE2) production, however, inhibitory effects on tumor necrosis factor-alpha (TNF-α) has not been reported. In this study, we evaluated the anti-inflammatory activity of SHT on inhibitory effects on TNF-α and PGE2 production and presented an analytical method for validation of SHT.
Experimental approach: Anti-inflammatory activity was evaluated by inhibitory activity on TNF-α and PGE2 production in RAW264.7 cells. The validated procedure was conducted according to ICH guidelines. The validated parameters were specificity/selectivity, linearity, range, the limit of detection (LOD), and limit of quantitation (LOQ).
Findings/Results: Ethanolic extract of SHT exerted inhibitory activity on PGE2 production in RAW264.7 cells with IC50 16.97 ± 1.16 μg/mL. Myristica frangrans seed extract showed the highest inhibitory activity on PGE2 production. Piper retrofractum extract showed the highest inhibitory activity on TNF-α production. For the HPLC method, all validated parameters complied with standard requirements. Each analyzed peak showed good selectivity with a baseline resolution greater than 1.51. The linearity of all compounds was > 0.999. The % recovery of all compounds was within 98.0-102.0%. The precision of all compounds was less than 2.0% CV.
Conclusion and implications: Ethanolic extracts of SHT possess anti-inflammatory activity by inhibition of TNF-α and PGE2 production in vitro. This study provides support for the traditional use of SHT. The validated results showed good specificity/selectivity, linearity, precision, and accuracy with appropriate LOD and LOQ. This study is the first report on the validation of the HPLC method of SHT for use as quality control of the SHT extract.

Keywords: Anti-inflammatory; HPLC; Method validation; PGE2; Sahastara; TNF-α


How to cite this article:
Kakatum N, Itharat A, Pipatrattanaseree W, Kanokkangsadal P, Davies NM. Validation of an HPLC method for quantification of anti-inflammatory markers in an ethanolic extract of Sahastara and its anti-inflammatory activity in vitro. Res Pharma Sci 2021;16:227-39

How to cite this URL:
Kakatum N, Itharat A, Pipatrattanaseree W, Kanokkangsadal P, Davies NM. Validation of an HPLC method for quantification of anti-inflammatory markers in an ethanolic extract of Sahastara and its anti-inflammatory activity in vitro. Res Pharma Sci [serial online] 2021 [cited 2021 Jun 18];16:227-39. Available from: https://www.rpsjournal.net/text.asp?2021/16/3/227/314821


  Introduction Top


Inflammation is a complex biological response protecting the body from harmful stimuli. Several cells and mediators are involved in the inflammatory process such as neutrophils, mast cells, and macrophages. Proinflammatory cytokines and inflammatory mediators such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and prostaglandin E2 (PGE2) are produced from these inflammatory cells during the inflammatory process. The over functioning of these mediators cause cell and tissue damage leading to muscle pain, and arthritis[1]. Non-steroidal anti-inflammatory drugs (NSAIDs) and steroidal drugs are utilized for the treatment of inflammation and pain. Patients with chronic inflammatory diseases such as rheumatoid arthritis and osteoarthritis need to be treated chronically. Long-term administration of steroids and NSAIDs can cause negative adverse events such as bleeding, indigestion, heart problems, and kidney toxicity[2]. Some patients select herbal medicines as alternative treatments. In traditional Thai medicine (TTM), several polyherbal remedies have been used for the management of inflammatory diseases such as Sahastara (SHT) and Lom-Am-Ma-Preuk remedies. These TTMs are prescribed according to different types of inflammatory diseases diagnosed by traditional Thai physicians[3].

SHT is a TTM categorised in the group of drugs for the treatment of musculoskeletal symptoms in the National List of Essential Medicines of Thailand. The remedy consists of 21 plant components. Piper nigrum (fruit), Plumbago indica (root), Terminalia chebula (fruit), Baliospermum montanum (root), Acorus calamus (root), Piper retrofractum (fruit), and Kleinhovia hospita (Bark) are the major ingredients. Each plant ingredient is combined in a different proportion according to [Table 1][4]. Previous pharmacological study reported that plant components in SHT remedy exerted anti-inflammatory activity by inhibiting the production of NO and cyclooxygenase 2 (COX-2) activity (IC50 2.81 and 16.97 μg/mL)[5]. Some plant components in SHT including Piper nigrum, Piper longum, Plumbago indica, Acorus calamus, and Terminalia chebula may have anti-inflammatory activity[5]. The anti-inflammatory mechanism of an ethanolic extract of SHT suggests anti-inflammatory activity via TNF-α and PGE2 production[6].
Table 1: Plant compositions of Sahasrara remedy in 1000 g

Click here to view


With regard to the phytochemical analysis of SHT, a quantitative analysis of chemical markers of SHT extract by HPLC has been reported by Kanokkangsadal and co-workers in a stability study of SHT extract. They determined only piperine which is a major component of Piper nigrum and in the SHT extract[7]. A previous study by Asasutjarit and co-workers also showed an HPLC method for the determination of piperine in a film-forming solution as a transdermal delivery system[6]. A previous study by Nuengchamnong and Ingkaninan reported an LC-MS/DPPH approach for the determination of antioxidative compounds in SHT, in which they analyzed hydrolyzable tannins[8]. All the previously published methods have requisite analytical validation data.

Thus, the objectives of this study were to investigate the anti-inflammatory activity by lipopolysaccharide (LPS)-stimulated TNF-α and PGE2 productions in macrophage RAW264.7 cells and to develop and validate an HPLC method for the determination of anti-inflammatory markers of SHT ethanolic extract. This study supported the traditional medical use of SHT and the validated method can be used as a quality control method for SHT extract and products.


  Materials and Methods Top


Plant materials and preparation of SHT extract

All plant materials were purchased from various sources, as shown in [Table 1]. The voucher specimens were deposited at the Herbarium of Southern Center of Thai Medicinal Plants at the Faculty of Pharmaceutical Science, Prince of Songkhla University, Songkhla province, Thailand and the voucher specimen IDs are included in [Table 1]. Plant materials were washed, dried, and cut into small pieces. Each plant component was weighed according to proportions shown in [Table 1] and mixed homogenously. The SHT remedy (1000 g) was macerated with 95% ethanol (3 L) for three days. The extract was filtered and evaporated at 45 °C under vacuum to dryness. The residue of SHT was then re-extracted two more times. All three extracts were combined and evaporated in a vacuum oven at 45 °C to dryness. The percentage yield of SHT was 8.44% w/w of the dried crude extract. The ethanolic extract of SHT was kept at -20 °C until used.

Chemicals and reagents

Gallic acid, ellagic acid, piperine, β-asarone (Merck, Darmstadt, Germany); plumbagin (Sigma-Aldrich, Seelze, Germany); acetonitrile and phosphoric acid (Labscan, Bangkok, Thailand); purified water was prepared by Milli Q® system from Millipore (Bedford, MA, USA); murine macrophage leukemia cell line (RAW 264.7: ATCC® TIB-71™; American Type Culture Collection, VA, USA); Roswell Park Memorial Institute medium 1640 (RPMI-1640), fetal bovine serum (FBS), penicillin-streptomycin (P/S), and 0.5% trypsin-EDTA (Gibco BRL Life Technologies, NY, USA); phosphate buffer saline (PBS; Amresco, Ohio, USA); dimethyl sulfoxide (DMSO; Fluka ,Munich, Germany); LPS and 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich Inc., MO, USA); PGE2 EIA kit (monoclonal; Cayman Chemical Company, MI, USA); mouse TNF-α Quantikine ELISA Kit (R&D Systems Inc., MN, USA).

Inhibitory effect on LPS-induced TNF-α production from RAW 264.7 cells

The inhibitory effect of SHT and its plant components on the release of TNF-α from RAW 264.7 cells was evaluated by using mouse TNF-α quantikine ELISA kit according to the method from Makchuchit et al.[9] and manufacturer’s instructions. In brief, RAW 264.7 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 10,000 U/mL penicillin, and 10,000 μg/mL streptomycin. The cells were maintained in an incubator at 37 °C in a humidified atmosphere containing 5% CO2. The cells were seeded in 96-well plates at a density of 1 × 105 cells/well and incubated in a humidified atmosphere containing 5% CO2 at 37 °C for 24 h. After that, the medium was replaced with fresh RPMI 1640 medium containing 5 ng/mL of LPS (100 μL/well) and treated with various concentrations of test samples (100 μL/well) and then the plate was incubated at 37 °C in 5% CO2 for 24 h. The stock solution of the test sample was prepared by dissolving the crude extract with DMSO to produce a stock solution at a concentration of 10 mg/mL. Then the stock solution was serially diluted by medium to produce a working solution of the test sample (0.2, 2, 20, 60, 100 μg/mL). The final concentration of DMSO in the treated cell was not more than 0.2% v/v. After 24 h incubation, the supernatant (cultured medium) was collected and the concentration of TNF-α was determined according to the instructions of the mouse TNF-α quantikine ELISA kit. The ELISA reaction was measured by a microplate reader (Bio Tek® Vermont, USA) at 405 nm. The inhibition (%) of TNF-α production was calculated by the following equation, and concentration causing 50% inhibition effect (IC50) values were calculated from the Prism program, (GraphPad prism 5,0, California, USA).



where, OD (sample) stands for optical density of sample; OD (control), optical density of diluent.

The viability of RAW 264.7 cells was determined by the MTT assay. After collecting the supernatant for determining TNF-α, 10 μL of MTT solution (5 mg/mL) was added to each well and incubated at 37 °C, 5% CO2, for 2 h. After that, the medium was removed and 100 μL of 0.04 M of hydrochloric acid (HCl) in isopropanol was added to dissolve the MTT formazan. The OD of the solution was measured at 570 nm. Cell survival (%) was calculated and demonstrated > 70% viability.

Inhibitory effect on LPS-induced PGE2 production from RAW 264.7 cell

The inhibition effect of SHT and its plant components on the release of PGE2 from RAW 264.7 cells was evaluated using a prostaglandin E2 EIA kit (monoclonal) according to the manufacturer’s instructions. In brief, cell culture and sample treatment protocols were conducted similar to the study protocol of anti-TNF-α described above except for the concentration of LPS. In this assay, the production of PGE2 from RAW264.7 was induced by LPS at a concentration of 5 μg/mL. The culture medium after sample treatment (0.1, 1, 10, 30 μg/mL) was collected and the amount of PGE2 production was determined by the PGE2 EIA kit, Indomethacin was used as a positive control. The inhibition (%) of PGE2 production was calculated by the following equation, and IC50 values [Table 2] were calculated from the Prism program.
Table 2: Inhibitory effects (IC50) on tumor necrosis factor-alpha and prostaglandin E2 production from RAW264.7 of the ethanolic extracts of Sahasthara and its plant components (n = 3)

Click here to view




where, OD (sample) stands for optical density of sample (treated in LPS-induced cells); OD (active), optical density of diluent (treated in LPS-induced cells); OD(inactive), optical density of diluent (treated in non-LPS-induced cells).

HPLC analysis

The analytical method for the determination of anti-inflammatory markers in SHT ethanolic extract was carried out by using an HPLC system (Shimadzu, Japan) consisting of a quaternary pump (LC20AD), an autosampler (SIL 20ACHT), a column oven (CT20A), and a diode array detector (SPD M20A). All markers were separated along a C18 HPLC column (5 μm, 4.6 mm × 250 mm; Phenomenex, USA) protecting by a C18 guard cartridge (5 μm, 4.6 mm × 3 mm; Phenomenex, USA). The mobile phase consisted of acetonitrile (A) and 0.1% v/v phosphoric acid in water (B). The gradient elution of mobile phase was programmed as the following: 0-20 min, 5% A:95% B; 21-49 min, 5% A:95% B - 50% A:50% B; 50-55 min, 50% A:50% B - 95% A:5%B; 55-60 min 5% A:95% B. The flow rate was set at 1.0 mL/min. The operating temperature was maintained at room temperature. Ten μL of sample and standard solutions were injected into the chromatographic system and the anti-inflammatory markers were detected at a wavelength of 256 nm.

Preparation of standard solutions

The anti-inflammatory markers of SHT extract analyzed in this study were gallic acid, ellagic acid, plumbagin, piperine and β-asarone. The stock solution of each standard was prepared in methanol at a concentration of 1 mg/mL. The working solutions of the mixed standard were prepared by diluting the stock solution of each standard in the same container and adjusting to the volume with methanol. The range of each compound is shown in [Table 3].
Table 3: Linearity, the limit of detection and limit of quantitation of gallic acid, ellagic acid, plumbagin, piperine, and β-asarone

Click here to view


Preparation of sample solutions.

The sample solution of ethanolic extract of SHT was prepared by dissolving an accurate amount of crude extract SHT in methanol to produce a sample solution at a concentration of 10 mg/mL of crude extract. The obtained sample solution was filtered through a 0.45 μm membrane filter before injection into the HPLC system.

Validation of the HPLC method

Validation of the analytical method for simultaneous quantification of anti-inflammatory markers: gallic acid, ellagic acid, plumbagin, piperine, and β-asarone in ethanolic extract of SHT was conducted according to the International Conference on Harmonization (ICH) guidelines, ICH Q2 (R1)[10]. The validated parameters in this study were specificity/selectivity, LOD, LOQ, linearity, range accuracy, and precision.

Specificity/selectivity

The specificity of the method was evaluated by comparing the chromatograms obtained from the analysis of sample solution, standard solution, and blank (methanol). The UV spectrum of the peaks in the chromatogram of the sample solution corresponded to the respective peaks of markers in the standard solution. The purity of each peak was determined by comparing the similarity pattern of UV spectrum at peak-start, peak-apex, and peak-end.

Linearity and range

The linearity of the method was performed by the coefficient of determination (r2) of the linear calibration curves. Each calibration curve of markers was constructed by six concentrations of the working standard solution containing gallic acid, ellagic acid, plumbagin, piperine, and β-asarone. The r2 of all curves were calculated.

Accuracy

The accuracy of the method performed is defined as the closeness of analytical results to the true value. Working standard solutions containing gallic acid, ellagic acid, plumbagin, piperine, and β-asarone were spiked into the SHT solution at three levels; low, medium and high concentrations. The spiked sample and un-spiked sample were analyzed by the HPLC method. Three replications of the spiked samples were analyzed in an analytical run over three consecutive analytical runs. The accuracy was shown as % recovery.

Precision

The precision of the analytical method performed is defined as the closeness of analytical results obtained from multiple analyses. The precision was conducted by analysis of standard solutions containing gallic acid, ellagic acid, plumbagin, piperine and β-asarone at low, medium, and high concentrations. The intra-run and inter-run precisions were performed as the coefficient of variation (CV) which were calculated from triplicate analyses in a run over three consecutive runs (6 days).

LOD and LOQ of the assay

The LOD and LOQ of the method were determined based on the visualization method. The standard solution was diluted and analyzed using the developed HPLC method. The LOD and LOQ of markers were calculated based on three times and ten times signal-to-noise ratio, respectively.


  Results Top


Anti-inflammatory activity of SHT ethanolic extract on LPS-induced TNF-α and PGE2 production in RAW264.7 cells.

The ethanolic extracts of SHT and its plant components were evaluated for inhibitory effects on LPS-induced TNF-α and PGE2 production in RAW264.7 cells compared to a positive control, indomethacin. As shown in [Table 2], SHT exerted potent inhibitory activity on PGE2 production with IC50 of 16.97 ± 1.16 μg/mL. However, SHT extract showed weak inhibitory activity on TNF-α production with an IC50 of more than 50 μg/mL.

With regard to plant components of SHT, the extract of seed of M. fragrans exerted the highest activity of inhibition of PGE2 production with IC50 of 16.99 ± 1.11 μg/mL following by P. nigrum (17.70 ± 1.43 μg/mL), and P. retrofractum (23.08 ± 1.79 μg/mL). All extracts exerted inhibitory activity less than indomethacin. For the inhibitory activity on TNF-α production, P. retrofractum showed the highest inhibitory activity with IC50 of 15.74 ± 2.50 μg/mL following with A. lancea (19.63 ± 1.13 μg/mL) and P.nigrum (20.74 ± 0.26 μg/mL).

Validation of HPLC method

Specificity/selectivity

The analytical method for the simultaneous determination of five anti-inflammatory compounds in SHT ethanol extract was developed and validated. As shown in [Figure 1], the chromatogram of the sample was compared to the chromatogram of standard solution and blank (methanol). The retention time of gallic acid, ellagic acid, plumbagin, piperine, and β-asarone in the sample solution were 10.27, 37.51, 50.18, 50.41, and 50.74 min, respectively. The analyzed peaks in the chromatogram of the sample solution corresponded to the respective standards in the chromatogram of the standard solution. Comparison of each peak in the chromatogram of the sample at peak-start, peak-apex, and peak-end position showed a similar pattern [Figure 2]. These results indicated the specificity and selectivity of this method.
Figure 1: The HPLC chromatogram of (A) methanol, (B) mixed standard solution, and (C) Sahasrara sample solution. X-axis is retention time (min) and Y-axis is intensity (milliabsorbance unit as mAU)

Click here to view
Figure 2: UV-spectrum of anti-inflammatory markers; (A and F) gallic acid, (B and G) ellagic acid, (C and H) plumbagin, (D and I) piperine, and (E and J) β-asarone. Panels A-E perform a comparative UV-spectrum of each marker’s peak in the sample solution to its respective standard. Panels F-J performs a comparative UV-spectrum of each marker in sample solution at peak-start, peak-apex, and peak-end. X-axis is the wavelength (nm) and Y-axis is absorptivity (milliabsorbance unit as mAU)

Click here to view


Linearity and ranges

The linearity of the developed method was determined by r2 calculated from the constructed calibration curves of each marker. As shown in [Table 3], the r2 values of gallic acid, ellagic acid, plumbagin, piperine, and β-asarone were 0.9999, 0.9997, 0.9999, 0.9998, 0.9991, respectively. The linear range of each marker was varied relating to the contents of the SHT extract and their absorptivity at a wavelength of 256 nm.

LODs and LOQs

LODs and LOQs of the markers were analyzed according to the visualization shown in [Table 3]. The LODs of gallic acid, ellagic acid, plumbagin, piperine, and β-asarone were 3.12, 0.625, 0.78, 0.9, 2.5 μg/mL, respectively. The LOQs were 6.25, 1.25, 1.56, 2.31, 5 μg/mL, respectively.

Accuracy and precision

The accuracy of the method was presented as % recovery of each compound shown in [Table 4]. The % recovery of all standards was within 99.09-101.88%. The precision of the analytical method, both intra- and inter-run, was performed as % CV which was less than 1.35.
Table 4: Accuracy of the analytical method for determination of gallic acid, ellagic acid, plumbagin, piperine and β-asarone in SHT extract

Click here to view


Content of anti-inflammatory markers from ethanolic extract of SHT remedy

The ethanolic extract of SHT remedy was analyzed for content using the developed and validated HPLC method. The results found the amount of gallic aid, ellagic acid, plumbagin, piperine, β-asarone were 18.26, 4.50, 11.48, 246.90, 104.30 mg/g, respectively.


  Discussion Top


Inflammation is a complex biological response protecting the body from harmful stimuli. Several cells and mediators are involved in the inflammatory process including pro-inflammatory cytokines, chemokines, and inflammatory immune cells[1]. PGE2 is an important inflammatory mediator produced from the COX pathway. It is the most abundant PGE produced in the body. PGE2 involves the classical signs of inflammation including redness, swelling, and pain[11]. TNF-α is an important pro-inflammatory cytokine expressed during the inflammatory process. It plays an important role in the immune system resulting in the pathophysiology of inflammatory diseases including arthritis and osteoarthritis[2].

SHT is a traditional Thai medicine approved by the Thailand National List of Essential Medicines. It has been indicated for the treatment of muscle and joint pain[4]. According to the traditional methods used, in this study, we investigated inhibitory effects of SHT and its components on PGE2 and TNF-α production in LPS-induced RAW264.7 macrophage cells. The ethanolic extract of SHT exerted anti-inflammatory activity via inhibition of PGE2 production from LPS-induced RAW 264.7 cells and weak activity on inhibition of TNF-α production. This study is the first report of SHT ethanol extract on in vitro inhibitory effects on PGE2 and TNF-α production. A previous study reported that the ethanolic extract of SHT exerted anti-inflammatory activity by inhibition of NO production in RAW264.7 cells with an IC50 of 2.56 μg/mL[4]. A clinical study by Pinsornsak et al. reported that oral 1000 mg SHT powdered (in capsule preparation), given 3 times daily for 28 days, significantly reduced pain and improved the symptoms of osteoarthritis patients[12]. There was no significant change in the blood chemistry of renal and liver functions in SHT treated group but the patients who took diclofenac showed significant increases in their aspartate aminotransferase, alanine transaminase, and alkaline phosphatase[12]. These results demonstrated the potential of SHT powder capsules for treating osteoarthritis patients. However, the ethanolic extract of SHT should be studied further for safety and efficacy in animal models prior to commencing clinical trials as an anti-inflammatory therapy in human.

With regard to plant components of SHT, P. nigrum and P. retrofractum exerted anti-inflammatory activity as measured with both PGE2 and TNF-α production. A. lancea showed inhibitory activity on TNF-α production. P. nigrum is the major ingredient of SHT with a proportion of 24% while P. retrofractum is 9.6%. For other major components, gallic acid and ellagic acid are the two anti-inflammatory compounds in Terminalia spp.[13], plumbagin is the major compound found in P. indica[14], piperine in Piper spp.[15], and β-asarone in A. calamus[16]. These compounds have been previously reported for their anti-inflammatory activities. Piperine inhibited the production of interleukin (IL6) and PGE2 in IL1β-stimulated fibroblast-like synovio-cytes[17],[18]. Ellagic acid and gallic acid inhibited the stimulation of COX-2 production in the LPS-induced RAW264.7 cells[19],[20]. Gallic acid also suppressed the release of IL-6, CCL7, and CXCL8, in IL-31- and IL-33-treated eosinophils-dermal fibroblasts co-culture[21]. β-asarone suppressed the production of TNF-α and IL-1β[22],[23]. Plumbagin inhibits NF-κB, resulting in a reduction of TNF-α, IL-1β, and IL-6, COX-2, and iNOS expression[24]. A previous study of Pinkaew and co-worker reported the clinal efficacy and safety on the treatment of osteoarthritis patients of Phyllanthus amarus cream. Gallic acid was the marker of this preparation. The results demonstrated that Phyllanthus amarus cream decreased symptoms of knee osteoarthritis as evaluated by the visual analog scale and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC)[25]. A previous clinical study by Schell et al. demonstrated that freeze-dried strawberry powder as a dietary supplement improved pain and inflammation in obese adults as evaluated by Intermittent and Constant Osteoarthritis Pain (ICOAP). Ellagic acid was a marker of the preparation and its plasma level was monitored[26]. A previous clinical study of a traditional Thai medicine named Benjakul on safety and efficacy in osteoarthritis patients demonstrated that Benjakul decreased the visual analog scale pain score and 100-meter walking times. Piperine and plumbagin are the chemical markers of Benjakul remedy[27]. Therefore, gallic acid, ellagic acid, plumbagin, piperine, and β-asarone were selected as the markers for anti-inflammatory activities in SHT.

The present study aimed at developing and validating of an HPLC method for the determination of the main anti-inflammatory constituents of an SHT extract. The method can be used as a quality control method for SHT extracts for further use as a traditional drug formulation in patients. However, there is no previous analytical method reported for quality control of these anti-inflammatory markers in SHT. This present study is the first study presenting validation results of an HPLC method for the determination of anti-inflammatory markers; gallic acid, ellagic acid, plumbagin, piperine and β-asarone.

A previous study from Kanokkangsadal and co-workers determined only the content of piperine in SHT ethanol extract[7]. Asasutjarit et al. also determined the content of piperine in film-forming containing SHT[6]. A previous study by Nuengchamnong and Ingkaninan analyzed the antioxidant compounds using an on-line LC-MS/DPPH approach which determined the hydrolysable tannins[8]. All previous HPLC methods did not report ICH validation results.

An HPLC method for simultaneous quantification of anti-inflammatory markers, gallic acid, ellagic acid, plumbagin, piperine, and β-asarone in SHT extract was developed and validated. In the present study, 0.1% v/v phosphoric acid was used as a component in the mobile phase for suppressing the ionization of phenolic compounds, such as gallic acid and ellagic acid, causing the reduction of peak tailing. The gradient elution was adjusted to separate peaks of piperine and β-asarone to obtain a peak resolution value of more than 1.5. The concentration range of the calibration curve of each marker was varied depending on the content of the markers in SHT extract. All markers were detected at a wavelength of 256 nm. The developed HPLC method showed good specificity for the determination of the anti-inflammatory markers in SHT ethanolic extract. The calibration curves showed good linearity with r2 > 0.999 at the analyzed concentration ranges. The LODs for all markers ranged between 0.625-3.12 μg/mL and the LOQs ranged between 1.25-6.25 μg/mL. The accuracy of each marker ranged between 99.55-101.35%. The intra-run and inter-run precisions presented as %CV were less than 1.35%. The method validation showed excellent results that can be used for quality control of the SHT extract.


  Conclusion Top


SHT is a traditional Thai medicine for muscle and joint pain. In this study, SHT possessed anti-inflammatory activity as measured by inhibition of PGE2 production in vitro. This study provided proof of anti-inflammatory activity supporting the traditional treatment of SHT. For quality control of the SHT ethanol extract, the developed HPLC method for simultaneous quantification of anti-inflammatory markers was validated. The validated results showed good selectivity, linearity, precision, and accuracy with appropriate LOD and LOQ. This study is the first report of validation of an HPLC method of SHT for use in quality control of the SHT extract and products.

Acknowledgements

The authors wish to thank the National Research Funding of Thailand under Thammasat University, Center of Excellence in Applied Thai Traditional Medicine Research (CEATMR) and Bualuang ASEAN Chair Professorship, Faculty of Medicine, Thammasat University, Khlong Luang, Pathum Thani, Thailand for funding for this research.

Conflict of interest statement

The authors declared no conflict of interest in this article.

Authors’ contribution

N. Kakatum conducted all the research. A. Itharat as the supervisor of N. Kakatum helped in the scope of research, the idea of the work, and participated in extraction, biological testing, chemical analyses, and interpretation of all data. A. Itharat, head of the Center of Excellence in Applied Thai Traditional Medicine Research (CEATMR), provided all funding and facilities. W. Pipatrattanaseree helped N. Kakatum for validation method of HPLC and writing the HPLC part. P. Kanokkangsadal assisted for training N. Kakatum on biological test. A. Itharat, W. Pipatrattanaseree and N. M. Davies wrote the article. N. M. Davies and A. Itharat helped in interpreting, revising, and editing the article.



 
  References Top

1.
Chen L, Deng H, Cui H, Fang J, Zuo Z, Deng J, et al. Inflammatory responses and inflammation-associated diseases in organs. Oncotarget. 2017;9(6):7204-7218. DOI: 10.18632/oncotarget.23208.  Back to cited text no. 1
    
2.
Bindu S, Mazumder S, Bandyopadhyay U. Non-steroidal anti-inflammatory drugs (NSAIDs) and organ damage: a current perspective. Biochem Pharmacol. 2020;180:114147,1-22. DOI: 10.1016/j.bcp.2020.114147.  Back to cited text no. 2
    
3.
Khunsopitbunnalak. Khum-Pee-Pad-Thai-PanBo-Lan. 2nd ed. Bangkok: Ministry Publishing; 1970. pp.267.  Back to cited text no. 3
    
4.
Announcement of the National Drug System Development Board on National List of Essential Medicines 2020. Royal Thai Government Gazette. 2020 [cited 2020 Dec 3];137:254D. Available from: http://www.ratchakitcha.soc.go.th/DATA/PDF/2563/E/254/T_0003.PDF.  Back to cited text no. 4
    
5.
Kakatum N, Jaiarree N, Makchucit S, Itharat A. Antioxidant and anti-inflammatory activities of Thai medicinal plants in Sahasthara remedy for muscle pain treatment. J Med Assoc Thai. 2012;95(Suppl 1):S120-S126.  Back to cited text no. 5
    
6.
Asasutjarit R, Sookdee P, Veeranondha S, Fuongfuchat A, Itharat A. Application of film-forming solution as a transdermal delivery system of piperine-rich herbal mixture extract for anti-inflammation. Heliyon. 2020;6(6):e04139,1-11. DOI: 10.1016/j.heliyon.2020.e04139.  Back to cited text no. 6
    
7.
Kanokkangsadal P, Wanichsetakul P, Itharat A. The stability of Sahastara remedy ethanolic extract used for application in clinical study. Thammasat Med J. 2018;18(4):519-527.  Back to cited text no. 7
    
8.
Nuengchamnong N, Ingkaninan K. An on-line LC-MS/ DPPH approach towards the quality control of antioxidative ingredients in Sahastara. Songklanakarin J Sci Technol. 2017;39(1): 123-129. DOI: 10.14456/sjst-psu.2017.14.  Back to cited text no. 8
    
9.
Makchuchit S, Rattarom R, Itharat A. The anti-allergic and anti-inflammatory effects of Benjakul extract (a Thai traditional medicine), its constituent plants and its some pure constituents using in vitro experiments. Biomed Pharmacother. 2017;89:1018-1026. DOI: 10.1016/j.biopha.2017.02.066.  Back to cited text no. 9
    
10.
ICH Harmonised Tripartite Guideline. Validation of analytical procedures: text and methodology Q2(R1). International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Geneva, 2005:1-13.  Back to cited text no. 10
    
11.
Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol. 2011;31(5):986-1000. DOI: 10.1161/ATVBAHA.110.207449.  Back to cited text no. 11
    
12.
Pinsornsak P, Kanokkangsadal P, Itharat A. The clinical efficacy and safety of the Sahastara remedy versus diclofenac in the treatment of osteoarthritis of the knee: a double-blind, randomized, and controlled trial. Evid Based Complement Alternat Med. 2015;2015:103046,1-8. DOI: 10.1155/2015/103046.  Back to cited text no. 12
    
13.
Tanaka M, Kishimoto Y, Sasaki M, Sato A, Kamiya T, Kondo K, et al. Terminalia bellirica (Gaertn.) Roxb. Extract and gallic acid attenuate LPS-induced inflammation and oxidative stress via MAPK/NFB and Akt/AMPK/Nrf2 pathways. Oxid Med Cell Longev. 2018;2018:9364364,1-15. DOI: 10.1155/2018/9364364.  Back to cited text no. 13
    
14.
Jaisi A, Sakunphueak A, Panichayupakaranant P. Increased production of plumbagin in Plumbago indica root cultures by gamma ray irradiation. Pharm Biol. 2013;51(8):1047-1051. DOI: 10.3109/13880209.2013.775163.  Back to cited text no. 14
    
15.
Meghwal M, Goswami TK. Piper nigrum and piperine: an update. Phytother Res. 2013;27(8):1121-1130. DOI: 10.1002/ptr.4972.  Back to cited text no. 15
    
16.
Das BK, Swamy AV, Koti BC, Gadad PC. Experimental evidence for use of Acorus calamus(asarone) for cancer chemoprevention. Heliyon. 2019;5(5):e01585,1-10. DOI: 10.1016/j.heliyon.2019.e01585.  Back to cited text no. 16
    
17.
Bang JS, Oh DH, Choi HM, Sur BJ, Lim SJ, Kim JY, et al. Anti-inflammatory and antiarthritic effects of piperine in human interleukin 1beta-stimulated fibroblast-like synoviocytes and in rat arthritis models. Arthritis Res Ther. 2009;11(2):R49,1-9. DOI: 10.1186/ar2662.  Back to cited text no. 17
    
18.
Liu Y, Yadev VR, Aggarwal BB, Nair MG. Inhibitory effects of black pepper (Piper nigrum) extracts and compounds on human tumor cell proliferation, cyclooxygenase enzymes, lipid peroxidation and nuclear transcription factor-kappa-B. Nat Prod Commun. 2010;5(8):1253-1257.  Back to cited text no. 18
    
19.
Corbett S, Daniel J, Drayton R, Field M, Steinhardt R, Garrett N. Evaluation of the anti-inflammatory effects of ellagic acid. J Perianesth Nurs. 2010;25(4):214-220. DOI: 10.1016/j.jopan.2010.05.011.  Back to cited text no. 19
    
20.
Tsang MS, Jiao D, Chan BC, Hon KL, Leung PC, Lau CB, et al. Anti-Inflammatory activities of pentaherbs formula, berberine, gallic acid and chlorogenic acid in atopic dermatitis-like skin inflammation. Molecules. 2016;21(4):519-539. DOI: 10.3390/molecules21040519.  Back to cited text no. 20
    
21.
BenSaad LA, Kim KH, Quah CC, Kim WR, Shahimi M. Anti-inflammatory potential of ellagic acid, gallic acid and punicalagin A&B isolated from Punica granatum. BMC Complement Altern Med. 2017;17:47-56. DOI: 10.1186/s12906-017-1555-0.  Back to cited text no. 21
    
22.
Kim DY, Lee SH, Kim WJ, Jiang J, Kim MK, Shin YK, et al. Inhibitory effects of Acorus calamus extracts on mast cell-dependent anaphylactic reactions using mast cell and mouse model. J Ethnopharmacol. 2012;141(1):526-529. DOI:10.1016/j.jep.2012.01.043.  Back to cited text no. 22
    
23.
Kim H, Han TH, Lee SG. Anti-inflammatory activity of a water extract of Acorus calamus L. leaves on keratinocyte HaCaT cells. J Ethnopharmacol. 2009;122(1):149-156. DOI: 10.1016/j.jep.2008.12.011.  Back to cited text no. 23
    
24.
Luo P, Wong YF, Ge L, Zhang ZF, Liu Y, Liu L, et al. Anti-inflammatory and analgesic effect of plumbagin through inhibition of nuclear factor-κB activation. J Pharmacol Exp Ther. 2010;335(3):735-742. DOI: 10.1124/jpet.110.170852.  Back to cited text no. 24
    
25.
Pinkaew D, Kiattisin K, Wonglangka K, Awoot P. Efficacy and safety of Phyllanthus amarus cream treatment in knee osteoarthritis. Open Sports Sci J. 2020;13:97-104. DOI: 10.2174/1875399X02013010097.  Back to cited text no. 25
    
26.
Schell J, Scofield RH, Barrett JR, Kurien BT, Betts N, Lyons TJ, et al. Strawberries improve pain and inflammation in obese adults with radiographic evidence of knee osteoarthritis. Nutrients. 2017;9(9):949-961. DOI: 10.3390/nu9090949.  Back to cited text no. 26
    
27.
Rachawat P, Pinsornsak P, Kanokkangsadal P, Itharat A. Clinical efficacy and safety of Benjakul remedy extract for treating primary osteoarthritis of knee compared with diclofenac: double blind, randomized controlled trial. Evid Based Complement Alternat Med. 2017;2017:9593580,1-9. DOI: 10.1155/2017/9593580.  Back to cited text no. 27
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
   Abstract
  Introduction
   Materials and Me...
  Results
  Discussion
  Conclusion
   References
   Article Figures
   Article Tables

 Article Access Statistics
    Viewed627    
    Printed8    
    Emailed0    
    PDF Downloaded96    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]