INTRODUCTION:

Analytical technologies and methods for the screening of metabolites occurring very fast. Nowadays analytical chemistry covers various ingenious field of study and out of which metabolomics, an analytical platform used for the large-scale and qualitative or quantitative analyses of metabolites of biological origin.

There have been various analytical technologies which have been utilized for metabolomic studies, among the various techniques like liquid chromatography, mass spectrometry, gas chromatography and Nuclear magnetic resonance (NMR), High performance Thin Layer chromatography has been the most powerful and suitable analytical technique in terms of its high flexibility, versatility, reproducibility and accuracy for getting rapid fingerprint region for a single chromatographic run for different samples¹. Chromatography is a non-destructive separation technique to separate individual component from a mixture². Quality of a drug is essential since everyone is consuming drug from the moment of birth and its quality can be estimated by analyzing it³. 55% of the available drugs used for treatments are from herbal origin and thus plants are most primary source of medicines for humans⁴. Standardization of herbal products in medicine is necessary as the demand increased in herbal formulations⁵. As the crude drugs have significant medicinal properties, so maintaining the quality in commercial product is essential⁶. To ensure the quality and potency of the herbal formulations, standardization in terms of raw materials and manufacturing process is essential⁷. Chemotherapeutic agents were having side effects to the normal healthy cells due to lack of organ specificity and minimum bioavailability of active drug, which leads to challenging aspects to the cancer management and treatment of cancer with minimum adverse effects or without it is a real challenge for reasearchers^8,9. In 1985 World Health Organization (WHO) estimated that 65% of the total world population depend on the plant-based medicine for the primary treatments¹⁰.

Erythrina stricta Roxb. (Papilionaceae) is a medium sized deciduous tree grown in various parts of China, Vietnam, Nepal, Thailand and India¹¹. Tetracyclic erythrinans and pterocarpans are mainly obtained from Erythrina plants belonging to the botanical family Leguminosae. Presence of various structures of erythrinan alkaloids and pterocarpans with their vast biological actions and their narrow distributions of their biologically active components attracted the interest of research of the plant^12,13,14,15. Erythrina genus contains more than 130 species that are widely available in the tropical and subtropical areas across the world. Ten species of Erythrina are available or grown in Yunnan province of China, which gives resources for evaluation of bioactivities of erythrinan alkaloids and pterocarpans and also systematic study on isolated new compounds¹⁶. The various parts of Erythrina stricta Roxb. have been reported to possess anticataract¹⁷, antimicrobial¹⁸, antioxidant¹¹, cardio-protective¹⁹, hyperuricaemia²⁰ and anti-inflammatory²¹.

MATERIALS AND METHODS:

Collection of plant materials:

Fresh bark of Eryjhrina stricta Roxb. was collected in the month of February-March (2018) from in and around of the city Aizawl, Mizoram and authenticated from Botanical survey of India, Eastern Region, Shillong, Meghalaya, India and specimen of herbarium was submitted in the RIPANS for future reference (Reference. BSI/ERC/TECH/2018-19/688).

Chemicals and Reagents:

All the chemicals and reagents were of analytical grades and purchased from Sigma-Aldrich, Germany.

Preparation of extracts:

Bark of the plant was cut in small pieces and dried under shade drying condition until fried completely. Weight was taken and extracted successively in soxhlet apparatus with series of solvents in increasing polarity such as Petroleum ether (ESPE), chloroform (ESC) and methanol (ESM). Materials were dried each time before extracting with the different solvent. Crude extracts were filtered using Whatman filter paper and concentrated using Rotary vacuum evaporator (IKA RV 10) and stored for further studies.

HPTLC fingerprint profiles of chloroform and methanol extracts:

TLC conditions:

HPTLC separation of ESC and ESM was performed on TLC plates pre-coated with silica gel 60 GF254 supported with aluminum sheet (20 x 10cm). The spotting device was Linomat V automatic sample applicator (CAMAG, Switzerland); the syringe (100µl, Hamilton); the developing chamber was CAMAG glass twin trough chamber (20 x 10cm), the densitometer comprises of CAMAG TLC scanner 3 (Slit dimension- 5 x 0.45mm and scanning speed- 20mm/sec), which was connected to WINCATS software and saturation time for mobile phase was 60 minutes.

Procedure:

Chloroform extract of Erythrina stricta Roxb. (ESC) and methanol extract of Erythrina stricta Roxb. (ESM) were selected for HPTLC fingerprint studies. Different solvent systems were tried with TLC and best resolution with solvent were selected as chloroform: methanol: acetic acid (9.5:0.7:1) and hexane: ethyl acetate: acetic acid (9:1:0.5) for ESM and ESC respectively. ESM and ESC were applied to a distance of 10mm. The plates were dried at room temperature. Plates were run in the development chamber saturated with mobile phase system for 45 minutes. After running up to 80% of the plates, plates were dried and scanned at 254nm with scanner. The R_fvalue of the developed chromatogram were recorded^22,23,24,25.

Cytotoxicity studies of Plant extracts:

Preparation of MTT solution:

MTT is soluble in ethanol (20mg/ml), water (10mg/ml) and culture media (5mg/ml) and buffered salt solutions. However, it is recommended using a 5mg/ml solution of MTT in PBS. Mix the content by vortexing or sonication and filter, sterilize solution after adding MTT.

MTT Assay:

Cell viability study was evaluated by the ability of the cells to break the tetrazolium salt MTT [3-(4,5-dimethylthiazol2-yl)-2,5- diphenyl tetrazolium bromide (Sigma, USA), by mitochondrial succinate dehydrogenase enzyme. Stock solution of each extracts were prepared by the solubilizing in dimethyl sulphoxide (DMSO) to give 1mg/ml concentration. From stock solution different concentrations (5, 10, 20, 40, 80, 100µg/ml) of ESC and ESM were prepared. A549 cells (NCCS, Pune) were applied and grown in 96-well microtitre for 24 hours. Different concentrations of the ESM and ESC extract were applied in the 96-well microtitre containing cells and incubated at 37⁰C in CO₂ environment for 48 hours. Positive control was doxorubicin (DOX), applied at a concentration of 1, 2, 4 and 8µg/ml. After 48 hours, supernatant were removed and 20µL of MTT (5mg/ml in DMEM) and again incubated for 4 hours. 100µL of DMSO was added to each well. Plate was placed on a plate shaker and gently shaken for 15 minutes to dissolve formazan crystals generated by proliferating cells. The measurement of optical density was performed using Spectramax M2 Microplate Reader (Molecular Diagnostic, Inc.) at a wavelength 570nm. Relative viability was estimated taking wells with non-treated cells as 100% control and standard deviation of the percentage cytotoxicity was calculated. 50% cytotoxic concentration of the extracts and positive control were determined and compared²⁶.

RESULTS:

Percentage yield of the plant extracts

Table 1. Percentage yield of Bark extract of Erythrina stricta Roxb.

Extract	Weight of Plant (a) (g)	Weight of extract (b) (g)	Percentage Yield(%) (b/a)x100
Petroleum Ether	1600	12.224	0.764
Chloroform	1585.24	54.547	3.44
Methanol	1527.31	90.247	5.90

HPTLC fingerprint profile of ESM and ESC.

Methanol extract of Erythrina stricta Roxb. (ESM):

HPTLC fingerprint profile of methanolic bark extract was performed after development of the plate in the Twin trough chamber saturated with solvent system Chloroform: Methanol: Glacial acetic acid (9.5:0.7:1) for 30 minutes. After scanning the plate at 254nm 12 different peaks were observed R_franging from 0.14 to 1.08. The peak diagram of the chromatogram is shown in Figure 1 below. The peak table of the chromatograms with different R_f value is shown in Table 2. Three dimensional diagrams of different peaks in the densitogram is also shown in figure 3

Figure 1. Peak diagram of methanolic extract of Erythrina stricta Roxb.

Figure 2 TLC plate seen at 254 nm of methanol extract of Erythrina stricta Roxb

Figure 3. Three dimensional (3D) representation of HPTLC chromatogram of methanolic extract of Erythrina stricta Roxb

Table 2. Peak table of methanolic extract of Erythrina stricta Roxb with R_f values

Peak	Start Rt	Start Height	Max Rt	Max Height	Max %	End Rt	End Height	Area	Area%	Assigned substance
1	0.02	3.3	0.08	606.5	28.51	0.14	209.1	22893.8	33.81	Unknown*
2	0.15	209.3	0.17	312.6	14.69	0.21	99.7	8259.3	12.20	Unknown*
3	0.30	88.1	0.31	88.8	4.18	0.36	70.1	2873.4	4.24	Unknown*
4	0.36	70.4	0.38	78.0	3.67	0.42	65.5	2770.0	4.09	Unknown*
5	0.42	65.8	0.43	67.4	3.17	0.49	47.1	2574.2	3.80	Unknown*
6	0.53	38.6	0.68	96.3	4.53	0.72	56.3	4241.8	6.26	Unknown*
7	0.63	38.6	0.68	96.3	4.53	0.72	56.3	4241.8	6.26	Unknown*
8	0.72	56.5	0.76	220.8	10.38	0.80	40.5	5786.7	8.56	Unknown*
9	0.80	40.8	0.86	116.6	5.48	0.88	105.0	3967.7	5.86	Unknown*
10	0.88	105.1	0.92	155.0	7.29	0.95	33.7	4998.8	7.38	Unknown*
11	0.96	34.3	0.97	41.0	1.93	0.99	0.2	585.2	0.86	Unknown*
12	1.01	0.0	1.06	208.1	9.78	1.08	24.1	3830.8	5.68	Unknown*

Table 3. Peak table of chloroform extract of Erythrin astricta Roxb withR_f values

Peak	Start Rt	Start Height	Max Rt	Max Height	Max %	End Rt	End Height	Area	Area%	Assigned substance
1	0.03	4.2	0.07	717.9	59.94	0.14	106.4	16670.9	55.81	Unknown*
2	0.15	106.6	0.16	112.0	9.35	0.23	23.1	3245.9	10.87	Unknown*
3	0.26	65.9	0.30	174.0	14.53	0.34	27.1	4211.3	14.10	Unknown*
4	0.34	27.4	0.37	38.8	3.24	0.38	34.0	829.0	2.77	Unknown*
5	0.42	28.8	0.45	79.9	6.67	0.51	12.5	2248.6	7.53	Unknown*
6	0.63	13.0	0.64	25.6	2.14	0.68	6.4	470.8	1.58	Unknown*
7	0.73	8.8	0.79	32.6	2.72	0.85	22.4	1837.5	6.15	Unknown*
8	0.91	10.1	0.93	16.9	1.41	0.96	3.7	358.8	1.20	Unknown*

Chloroform extract of Erythrina stricta Roxb. (ESC):

HPTLC fingerprint profile of chloroform bark extract was performed after development of the plate in the Twin trough chamber saturated with solvent system Hexane: Ethyl acetate: Glacial acetic acid (9:1:0.5) for 30 minutes. After scanning the plate at 254 nm 8 different peaks were observed R_f ranging from 0.14 to 0.96. The peak diagram of the chromatogram is shown in Figure 4. below. The peak table of the chromatograms with different R_f value is shown in Table 3. Three dimensional diagrams of different peaks in the densitogram is also shown in Figure-6.

Figure 4. Peak diagram of chloroform extract of Erythrina stricta Roxb.

Figure 5. TLC plate seen at 254 nm of chloroform extract of Erythrina stricta Roxb.

Figure 6. Three dimensional (3D) representation of HPTLC chromatogram of chloroform extract of Erythrina stricta Roxb.

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay

Table 4. The percentage cytotoxicity of A549 cell lines against ESM and ESM

Conc. (µg/ ml)	ESM		ESC		DOX
Conc. (µg/ ml)	Cytotoxicity (%)	STD	Cytotoxicity (%)	STD	Cytotoxicity (%)	STD
5(1)	-4.01	1.95	7.19	0.47	19.03	1.68
10(2)	13.52	0.53	26.82	1.35	43.18	2.34
20(4)	17.92	2.03	39.65	4.36	59.35	0.21
40(8)	24.75	2.24	55.42	2.77	72.47	0.06
80	35.38	1.47	56.54	0.64
100	44.99	2.36	68.56	0.92
IC50	135.5		39.99		3.73

Figure 7. The graph represents the percentage cytotoxicity of ESM and ESC against A549 cell lines. Parenthesis () indicates concentration of doxorubicin. N=3

Cytotoxicity of ESM and ESC were evaluated against A549 cell line and it was found that all the extract are active against A549 cell line. IC₅₀ values were calculated and shown in the Table 4. Comparative cytotoxicity of ESC, ESM and Doxorubicin (Dox) were shown in Figure 7.

DISCUSSION:

HPTLC is nowadays becoming a day-to-day analytical tool for its various advantages for its minimum clean up and low cost operation²⁷. HPTLC fingerprint profile of Erythrina stricta Roxb. for both methanol and chloroform extracts showed good number of peaks in the chromatogram which suggested the presence of different phytoconstitutents. Extractions and characterization of plant active metabolites leads to the discovery of new drugs with better medicinal values. Extracts from herbal origin can give synergistic effect to the human body or can also reduce the side effects to some extent. Herbal phytoconstituents are much safer than the synthetic drugs and also provide protection from diseases caused by free radicals and also protect from cell injury. Phytochemicals comprises of various primary and secondary metabolites obtained from plants. HPTLC and HPLC are found to be important tools for the evaluation, characterization and authentication of herbal drugs. The species of Erythrina were evaluated for HPTLC fingerprint for their proper authentication and evaluation of phytochemicals present in the plants²⁸.

Till now most of the cancer treatment is based on chemotherapy. Chemotherapeutics agents are having side effects. In recent years, the researchers shown interest in exploration of anticancer drugs from natural origin. In this study, Erythrina stricta Roxb., methanol and chloroform extracts were screened for in vitro cytotoxicity activity against A549 cell line (Lung cancer) using MTT study and compared against standard doxorubicin(Figure 7). IC₅₀ values were calculated and found 39.99 µg/mL and135.5 µg/mL for ESC and ESM against the standard doxorubicin (IC₅₀ = 3.73 µg/mL) (Table 4). Among these ESC have significant IC₅₀ value. IC₅₀ concentration corresponds to the death of 50% cells by the plant extracts. Thus these plant extracts reduce the cell viability by exerting cytotoxic effect to the A549 cells²⁹.

CONCLUSION:

Plants are very important source of various phytochemicals. Characterization and authentication of these phytochemicals is necessary for the proper identification and elimination of chances of adulteration for quality control purpose. In this study, HPTLC fingerprint profile of bark extract of Erythrina stricta Roxb. provides an important identification tool for this plant which can be used for future reference. The study of cytotoxicity by MTT assay reveals that bark extract of Erythrina stricta Roxb. possesses significant cytotoxic effect for A549 cell line (Lung cancer). The researcher can further isolate the lead molecule responsible for the anticancer activity and study the molecular pathway responsible for the cytotoxicity activity.

CONFLICT OF INTEREST:

We declare we have no conflict of interest.

ACKNOWLEDGEMENTS:

The authors would like to thank Dr. H. Lalhlenmawia, HoD, Department of Pharmacy, Dr. Chawngthanliana, Director, Regional Institute of Paramedical and Nursing Sciences (RIPANS), Aizawl and Prof. N. Senthil Kumar, Department of Biotechnology, Mizoram University for providing necessary facilities for carrying out the research work.

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Received on 22.06.2021 Modified on 09.10.2021

Research J. Pharm. and Tech 2022; 15(10):4451-4456.

DOI: 10.52711/0974-360X.2022.00746