Reverse Phase High-Performance Liquid Chromatographic Estimation and In vitro Cytotoxicity of Boswellic Acids on A-375 Melanoma Cancer Cell lines

 

Neeta, Harish Dureja*

Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak-124001 India

*Corresponding Author E-mail: harishdureja@gmail.com,

 

ABSTRACT:

The present investigation was aimed at extraction, characterization and in vitro cytotoxic effects of boswellic acids (KBA and AKBA) against A-375 Melanoma Cancer Cell lines. Methanolic extract of Boswellia serrata was prepared and gradient Reverse Phase High-Performance Liquid Chromatographic (RP-HPLC) method was used for the identification and quantification of boswellic acids in the gum resin extract. Presence of boswellic acids in the extract was further confirmed by TLC, FTIR and DSC. In vitro cytotoxicity of the extract was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay against A-375 Melanoma Cancer cells  Quantitative estimation of boswellic acids by RP-HPLC method was found to be around 40.6 mg/gram and 32.3 mg/gram for KBA and AKBA, respectively. MTT assay showed cytotoxic potential of boswellic acids against melanoma cell lines with IC50  of 254.9 µg/mL with significant cytotoxic effects.

 

KEYWORDS: Boswellic acids; TLC; RP-HPLC; FTIR; MTT assay.

 

 


INTRODUCTION:

Every year incidences of melanoma skin cancer tend to be increasing worldwide. Melanoma is malignant type tumor derived from melanocytes cells and incident of melanoma carcinoma in the United States and other developed countries varies among men and women which are fifth and seventh most common cancer type respectively.[1] Skin is the most common site for development of melanoma, even though primary melanoma develops in oral and nasal mucosa, vulval, anorectal mucosa, eye, other gastrointestinal mucosa.

 

Different risk factors linked with development of melanomas includes family history of melanoma, fair complexion, sensitivity of skin to sunburn different types of naevi (dysplastic naevi and giant congenital melanocytic naevi), a history of non-melanoma type skin cancers and most frequently the immune deficient persons. [2] Development of melanoma have been associated with numerous molecular events, revealed from various genomic and proteomic studies. [3] Many approaches have been made for the management of cancer disease because of its complexity; lack of organ specificity with minimum bioavailability of active agent’s leading to higher toxicity to normal and healthy cells of the body are major side effects of chemotherapeutic agents. For reduction of these constraints and side effects associated with cancer therapy, there is utmost requirement to design and fabricate therapeutic modalities with minimum or no side effects to healthy tissues. Utilization of naturally derived dietary phyto chemicals and herbal drug products is an effective approach to circumvent the side effects of chemotherapy. [4] Drug molecules derived from natural sources have offered the significant attainment in the chemotherapy for the treatment of various diseases. Boswellia serrata of Burseraceae family is deciduous middle sized plant, mostly found in tropical areas of Asian and African countries especially in dry forests of Gujarat, Madhya Pradesh, Assam, Bihar and Orissa in India. [4-6] Main constituents of the Boswellia serrata plants are Boswellic acids (BAs) belonging to the ursane group and Indian olibanum, Salai guggal, white guggal are commonly used synonyms of this plant. [7-8] Higher terpenoids constitute the major fraction of about 25-35% of gum resin which constitutes of four important BAs: α-boswellic acid, β-boswellic acid, 11-keto-β- boswellic acid (KBA), and 3-acetyl-11-keto-β-boswellic acid (AKBA) for their anti-inflammatory [7], anti-arthritic [9] anti-asthmatic [10] anti-proliferative [11] and anticancer activities. [12] Boswellic acid consists of pentacyclic ring skeleton which is essential for anti-topoisomerase activity revealed by various structural activity relationship studies. [13] The present study aimed with the extraction of BAs from gum resin of plant Boswellia serrata, qualitative estimation by thin layer chromatography and standardization by using RP-HPLC method for the estimation of boswellic acids by standard marker compounds. In addition, this study also deals with the characterization of BAs by Fourier Transform Infra red spectroscopy for structural identification. In vitro cytotoxic efficacy of the extract was also evaluated against A-375 Melanoma Cancer Cells.

 

MATERIALS AND METHODS:

Plant Material:

Gum resin of plant Boswellia serrata was procured from the local market and authenticated from National institute of science communication and information resources (NISCAIR) by Dr. H.B. Singh, Chief Scientist & Head, Raw Materials Herbarium & Museum (RHMD), NISCAIR.

 

Cell lines and cell culture:

A - 375 Melanoma Cancer cell lines were procured from NCCS (National Centre for Cell Science India). Cell lines were cultured in Dulbeco’s modified Eagle’s medium (DMEM-D2429) (Sigma Aldrich, Missouri, USA) deficient of folic acid and vitamin B12. Growth medium was supplemented with 10% Fetal Bovine Serum (Gibco, Invitrogen) and 1% Antibiotic - Antimycotic 100 solution (Thermofisher Scientific).

 

Chemicals and reagents:

Standard biomarkers KBA and AKBA for standardization of boswellic acids were procured from Sigma-Aldrich, Germany. HPLC grades Acetonitrile, Methanol, Phosphoric acid were purchased from Avantor Performance Material, Faridabad. All the other chemicals used including the solvents were of analytical grade.

 

Instruments:

Chromatographic standardization was done using Thermo Scientific, Ultimate 3000 series, comprising of a C18 column (4.6 mm× 250 mm, 5-micron particle size) including Dionex ultimate 3000 pump with an auto-sampler and diode array detector. Data acquisition and integration were controlled by chrome­leon software; version 6.80 SR. FT-IR Alpha Bruker 1206 0280, Germany, DSC Q 10 V 9.9, US, Ultracentrifuge 3-30KS, Sigma Laborgentrifugen, D-37520, Germany, Water bath sonicator, Bandelin, Germany, Bruker Avance II 400 MHz.

 

Extraction of boswellic acids from gum resin:

Gum resins of boswellic acids crushed into the smaller size and soaked in methanol for 12 h. Resulting solution was filtered and the filtrate was concentrated to obtain a reddish brown mass. A 3% w/v potassium hydroxide solution was used to basify the mass and continuous stirring was performed to formulate a uniform emulsion. Dichloromethane was used for separation of the lower solvent layer from this emulsion. BAs are found to be present in the upper layer which was then acidified with dilute hydrochloric acid to precipitate the acid content. Centrifugation was performed for the separation of precipitated acids and these acids were washed several times with distilled water. The light yellow powder obtained after drying was subjected to particle size analysis. [14]

 

Thin Layer Chromatography (TLC):

TLC is a simple chromatographic technique used for the separation of a number of components present in a given mixture by identification of a compound in a mixture with their respective retention factor (Rf) value. It is particularly also valuable for qualitative determination of small amounts of impurities. The finger print profile of the plant is easier to study based on the separation of colored bands, number of bands and Rf values. 10 mg of the powdered extract was dissolved in 5 mL of methanol. Sonication was done for 10 min and mixture was vigorously shaken four times and diluted (up to 10 mL) with methanol. Centrifugation was carried out at 5000 rpm to obtain the clear solution. The reference solution of standard marker compounds was prepared by addition of 2 mg of KBA and AKBA in 20 ml of methanol. Mobile phase consists of anhydrous formic acid, heptanes, ethyl acetate, toluene in the ratio of 3:10:20:80 v/v/v/v. 10 µl sample and standard markers were applied on TLC plate and developed in a pre-saturated glass chamber, dried and examined under ultraviolet light at 254 nm. Rf was determined for both sample and standard solutions by observing spots on TLC plates under UV trans-illuminator at 254 nm. Reference Rf value occurs between 0-1. [15-16]

 

Fourier Transform Infrared spectroscopy (FTIR):

FTIR is one of the commonly used analytical techniques for the identification and characterization of unknown compounds. This technique is based on the principle that each and every compound has specific absorption in the IR region and after absorbing these radiations, atoms present in the compounds undergo stretching and bending vibrations and leads to the generation of absorption spectra. The presence of different functional groups in a compound, every compound gives different and unique spectrum; therefore, FTIR is preferred analytical approach for the qualitative analysis. Boswellia extract was scanned on FTIR Alpha Bruker 1206 0280, Germany by using KBR pellets. [17-19]

 

Differential Scanning Calorimetry (DSC):

In order to observe crystalline or amorphous nature of the active drug, DSC is the most frequently used technique. The physical state of the extract was observed by DSC instrument (DSC Q10 V9.9., USA) by calibration with Indium as standard. An analysis was carried with respect to the reference as empty pan and samples in sealed aluminium pan under nitrogen atmosphere (60 ml/min) and heating at the temperature range of 30şC to 300şC. [20]

 

RP-HPLC Estimation of Boswellic acids:

Standard stock and test solutions:

The stock solutions of both the standards were prepared by dissolving accurately weighed 1 mg of each of KBA AKBA standards in 20 ml of HPLC grade methanol. Further dilutions were made with methanol to achieve a final concentration of 50 µg/mL. Stock solutions for the test sample was prepared by dissolution of 100 mg of extract in 10 ml of methanol and sonicated for 10 minutes. After centrifugation, a clear solution was further diluted with a mixture of mobile phase A and mobile phase B to get the final concentration of 50 µg/mL.

 

Chromatographic conditions:

Binary gradient program (Table 1) was used for the quantitative estimation of KBA and AKBA. The column used was octadecylsilane silica gel C-18 250 x 4.6 mm; 5 µm particles. The mobile phase A was a blend of 0.1: 99.9 v/v (Phosphoric acid: Water) and mobile phase B was having compositions of 0.1 : 99.9 v/v (Phosphoric acid: Acetonitrile). 0.45 µm membrane filters were used for the filtration of mobile phases with a run time of 8 minutes for KBA and 12 minutes for AKBA followed by scanning at 250 nm using diode array detector. 20 µl of the sample was injected and percentage content of KBA and AKBA was determined in the extract. Optimum HPLC conditions are represented in Table 2. The content of the individual boswellic acid was determined along with total boswellic acids content in Boswellia serrata. [15]

 

Table 1: Binary gradient program for HPLC

S. No

Time (min)

Mobile phase A (percent v/v)

Mobile phase B (percent v/v)

1.

0-12

16 -6

84-94

2.

12.5-13.5

6-0

94-100

3.

13.5-28

0

100

 

Table 2: Chromatographic conditions for HPLC analysis

Parameter

Chromatographic conditions

Instrument

Thermo Scientific, Ultimate 3000 series, Germany

Column

C -18 (250X4.6 mm; 5µm)

Detector

Diode Array Detector

Diluents

Mobile phase

Solvent system

Binary gradient system

Mobile phase A: 0.1:99.9 v/v (Phosphoric acid:Water)

Mobile phase B: 0.1:99.9 v/v (Phosphoric acid:Acetonitrile)

Flow rate

1.0 mL/min

Detection wave length UV

250 nm

Injection volume

20 µl

Total Run time

28 min

Retention time

 ~8 min KBA, ~12 min AKBA respectively

 

MTT Assay:

In vitro cytotoxicity of gum resin extract was evaluated by MTT assay based on colorimetric or spectro photometric determination. DMEM culture medium supplemented with 10% FBS and 1% Antibiotic Antimycotic 100X solution were used for cell lines culture. The cells were seeded at a density of approximately 5×103cells/well in a 96-well flat-bottom micro plate and maintained at 370C in 95% humidity and 5% CO2 for overnight. Different concentration (10, 20, 40, 80, 160, 320 µg/mL) of samples was treated. The cells were incubated for another 48 hours. The cells in well were washed twice with phosphate buffer solution and 20 µL of the MTT staining solution was added to each well and plate was incubated at 370C. After 4h, 100 µL of di- methyl sulfoxide (DMSO) was added to each well and absorbance was recorded at 570 nm using ELISA plate reader (Enzyme Linked Immunosorbent Assay). In order to calculate inhibitory concentration50 (IC50) which is representation of concentration of sample necessary to inhibit 50% of cells of; regression curve was constructed by plotting percentage viability against concentration.[21]

 

RESULTS:

The extraction of BAs was carried out by using methanol and its active components in gum resin extract were determined. The percentage extractive value was found to be 38.41% w/w.

 

Identification of KBA and AKBA by Thin Layer Chromatography:

TLC analysis was performed for identification of both KBA and AKBA in the gum resin extract and presence of test compounds were confirmed by used TLC method as shown in Figure 1.

 

FTIR and DSC Analysis:

The presence of BAs in the extract was further confirmed by analysis of structural composition using FTIR technique The FTIR spectra of obtained sample showed peaks at 1699 cm-1 for C=O stretching of aryl acid, 1648 cyclic ketone, 1453 cm-1 for CH2 scissoring of cyclohexane and 1238 cm-1 for C-Co-C skeleton alkyl ketone. The peak observed at 2942 cm-1 is assigned for OH stretching of carboxylic acid, 3442 cm-1 for OH stretching (bonded) observed when steric hinderance presents polymeric association and 1022 cm-1 and 984 cm-1 for ring stretching of cyclohexane. Various peaks are depicted in Figure 2 which revealed the presence of BAs in the gum extract. DSC thermogram showed an endothermic peak for KBA & AKBA at 168°C and 275 oC respectively corresponding to their reported melting point (Figure 3).

 

RP-HPLC quantitative estimation of Bas:

Qualitatively TLC showed the presence of BAs by spot analysis and Rf values of both KBA and AKBA were compared. The Presence of BAs (Qualitatively and quantitatively) was further confirmed by RP-HPLC chromatographic fingerprinting analysis by comparison of HPLC chromatogram retention time of peaks of KBA and AKBA test with chromatogram obtained from marker compounds (Figures 4-5). Reported retention time for KBA and AKBA was ~8 min and 12, min respectively. Retention time peaks for both KBA and AKBA were corresponded to their reported retention time 7.92 min and 12.36, min respectively.


 

 

Figure 1: Identification of Boswellic acids by TLC analysis: a) standard of KBA b)standard of AKBA c) Boswellia extract

 

 

Figure 2: FTIR spectra of Boswellia serrata extract

 

Figure 3: DSC thermogram of Boswellia extract

 

 

Figure 4: HPLC chromatogram of Standard (KBA and AKBA)

 

 

Figure 5: HPLC chromatogram of Boswellia extract (KBA and AKBA)


MTT Assay:

Cytotoxic effects of BAs were evaluated by MTT assay using the melanoma A-375 cancer cell lines based on colorimetric estimation of reduced MTT by mitochondrial succinate dehydrogenase enzyme. The range of concentrations of extract was 10 to 360 μg/ml and MTT results are shown in Table 3 which revealed concentration dependent cytotoxic effects of extract Figure 6(a). Regression graph was plotted for calculation of Inhibitory Concentration50 (IC50)  as represented in Figure 6(b) and IC50 value was found to be 254.9 µg/mL. MTT is only step used for investigation of in vitro cytotoxicity of the samples against cancer cell lines and give indications that the test samples may have the anticancer potential which can be further confirmed by other assays.

 

Table 3: Percentage cell viability at different concentrations against melanoma cancer cell

Test Sample Concentration μg/mL

Cell viability (%)

 

 

 

IC50 254.9 μg/mL

10

97.12±0.85

20

88.39±0.74

40

81.46±0.82

80

71.27±1

160

64.41±1.1

320

40.92±0.6

 

 

(a)

 

 

(b)

Figure 6: a) Percentage viability of cancerous melanoma cells (b) Regression graph for IC50 value

DISCUSSION:

Different analytical techniques were used for identification and quantitative estimation of BAs present in the methanolic extract of Boswellia serrata. Two main BAs (KBA and AKBA) are mainly responsible for different pharmacological activities like anti-inflammatory, anticancer and anti-arthritic etc. In the present study extracted BAs were identified by TLC where Rf values of standard marker compounds were at nearly about 0.39 for KBA and 0.42 for AKBA and approximately equivalent Rf  values and  intensity of bands confirmed the presence of KBA and AKBA in plant extract. Utilization of phyto chemicals fingerprinting by spectroscopy provide valuable information about structural identification of different compounds. Many research workers revealed the FTIR spectrum as an effective tool for differentiating, classifying and discriminating closely related compounds and mixture of compounds present in the plant extracts.  FTIR spectrometry was used to find out the presence of BAs in the extract. Various peaks shown in the spectrum revealed the presence of BAs in the gum resin extract. Similar work was also reported by Mehta et al., in IR spectra of methanolic extract of Boswellia serrata. [7] Further confirmation of KBA and AKBA in the extract was confirmed by DSC analysis. DSC thermogram showed an endothermic peak for KBA & AKBA at 168°C and 275 oC respectively corresponding to their reported melting point. [22] The melting point of the sample is almost similar to the reported value confirmed the presence of BAs. Herbal formulations being multi component system lack adequate analytical methods ascertain their quality and consistency. A concept of simultaneous estimation of more than one biologically active phyto constituents in a single set of analytical method is found to be beneficial.  The used RP-HPLC method in the current study was found to be accurate and give corresponding retention time peaks for KBA and AKBA for standard (~8 min and 12, min respectively) and test sample (7.92 min and 12.36, min respectively). Cancer disease is still increasing worldwide in spite of advancements in its diagnosis and treatment. Millions of people in all age group are affected by different types of cancers across the world and melanoma cancer is one of them. Currently various plant based anticancer based drugs are under investigation that target signaling as well as epigenetic pathways that can cause cancer. Therefore, it becomes necessary to isolate and investigate new anticancer agents derived from plant sources which are effective on growth inhibition of cancer. The cytotoxic and anticancer effects of BAs have been intensively studied and well documented in the last decade in both cell culture studies and animal models. In vitro cytotoxic effects of BAs were evaluated by MTT assay using the melanoma A-375 cancer cell line. MTT assay results revealed concentration dependent cytotoxic effects of extract and IC50 value was found to be 254.9 µg/mL. As reported previously by uthaman et al. [19]  MTT is only step used for investigation of in vitro cytotoxicity of the samples against cancer cell lines and give indications that the test samples may have the anticancer potential which can be further confirmed by other assays.

 

CONCLUSION:

Successfully extraction and identification by TLC and effectively RP-HPLC method were utilized for significant quantification of the percentage content of BAs in Boswellia serrata. Effective characterization of BAs was achieved by analyzing FTIR spectrum and DSC thermogram. The more marked inhibition of cell growth was obtained at higher concentration of BAs on the melanoma cancer cells and MTT assay proved to be fast tool for traditionally important medicinal plant proved to be an effective cytotoxic agent. Extraction, identification, and characterization of BAs will be helpful for the standardization of herbal formulations containing BAs.

 

CONFLICT OF INTEREST:

Authors have no conflict of interest

 

REFERENCES:

1.     Niezgoda A, Niezgoda P and Czajkowski R. Novel approaches to treatment of advanced melanoma: A review on targeted therapy and immunotherapy. BioMed Research International. 2015; 1-16.

2.     Perera E, Neiraja G, Ross J and Rodney S. Malignant melanoma. Healthcare. 2014; 2(1): 1-19.

3.     Miller AJ and Mihm MC. Melanoma. New England Journal of Medicine. 2006; 355: 51-65.

4.     Neeta and Dureja H. Role of Boswellic acids in cancer treatment. Journal of Medical Sciences. 2014; 14(6-8): 261-269.

5.     Eichhorn T, Greten HJ and Efferth T. Molecular determinants of the response of tumor cells to Boswellic acids. Pharmaceuticals. 2011; 4(8): 1171-1182.

6.     Upaganlawar A and Ghule B. Pharmacological Activities of Boswellia serrata Roxb. - Mini Review. Ethnobotanical Leaflets. 2009; 13(6): 766-774.

7.     Mehta M, Dureja H and Garg M. Development and optimization of boswellic acid loaded proniosomal gel. Drug Delivery. 2015; 23(8): 3072-3081.

8.     Shah BA, Qazi GN and Taneja SC. Boswellia acids: A group of medicinally important compounds. Natural Product Reports. 2009; 26(1): 72-89.

9.     Sharma MLBani S and Singh GB. Anti-arthritic activity of boswellic acids in bovine serum albumin (BSA)-induced arthritis. International Journal of Immunopharmacology. 1989; 119(6): 647-652.

10.  Pungle et al. Immunomodulatory activity of boswellic acids of Boswellia serrata Roxb. Indian J Experimental Biology. 2003; 41(12): 1460-1462.

11.  Liu JJ, Huang B and Hooi SC. Acetyl-keto-β-boswellic acid inhibits cellular proliferation through a p21-dependent pathway in colon cancer cells. British Journal of Pharmacology. 2006; 148(8): 1099-1107.

12.  Hostanska K, Daum G and Saller R. Cytostatic and apoptosis-inducing activity of boswellic acids toward malignant cell lines in vitro. Anticancer Research. 2002; 22(5): 2853-2862.

13.  Bhushan et al. A triterpenediol from Boswellia serrata induces apoptosis through both the intrinsic and extrinsic apoptotic pathways in human leukemia HL-60 cells. Apoptosis. 2007; 12(10): 1911-1926.

14.  Ali et al. Evaluation of systemic administration of Boswellia papyrifera extracts on spatial memory   retention in male rats. Journal of Natural Medicine. 2011; 65(3-4): 519-25.

15.  British Pharmacopeia. Indian Frankincense. (Ph. Eur. monograph 2310). 2012.

16.  Vani et al. Identification and chemical characterization of Azadirachta indica leaf extracts through thin layer chromatography. International Journal of Research in Engineering and Technology. 2016; 5(2): 117-122.

17.  Bunaciu AA, Aboul-Enein HY and Fleschin S. Recent applications of fourier transform infrared spectrophotometry in herbal medicine analysis. Applied Spectroscopy Reviews. 2011; 46(4): 251-260.

18.  Chanda S, Baravalia Y and Nagani K. Spectral analysis of methanol extract of Cissus quadrangularis L. stem and its fractions. Journal of Pharmacognosy and Phytochemistry. 2013; 2(4): 149-157.

19.  Uthaman S, Snima KS and Annapoorna M. Novel boswellic acids nanoparticles induces cell death in prostate cancer cells. Journal of Natural Products. 2012; 5: 100-108.

20.  Bairwa K and Jachak SM. Development and optimization of 3-Acetyl-11-keto-β-boswellic acid loaded polylactic- co-glycolic acid-nanoparticles with enhanced oral bioavailability and in-vivo anti-inflammatory activity in rats. Journal of Pharmacy and Pharmacology. 2015; 67(9): 1188-1197.

21.  Shah et al. Molecular and cellular effects of vitamine B12 forms on human trophoblast cells in presence of excessive folate. Biomed Pharmacotherapy. 2016; 84: 526-534.

22.  Siddiqui MZ. Boswellia serrata, A potential anti-inflammatory agent: An overview. Indian Journal of Pharmaceutical Sciences. 2011.

 

 

 

 

 

 

 

 

 

Received on 24.10.2017          Accepted on 11.02.2018        

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(1): 13-19.

DOI:  10.5958/2231-5675.2018.00003.0