Bio Active Compound Analysis of Croton scabiosus Bedd: By HPTLC, GC-MS and Evaluation of Anthelmintic Activity and Anticancer Potential on Lung (A549) and Breast (MCF-7) Cancer Cell Lines

Croton scabiosus Bedd. (Euphorbiaceae) is an endemic plant of Andhra Pradesh, India. Local communities use the verdant parts of this plant as an antidote to treat snake bites and as a remedy to heal leucorrhoea in the form of decoction. In the present study the author found to have a perceptible range of alkaloids, coumarins, glycosides, flavonoids, steroids and gallic tannins as secondary metabolites by phytochemical and spectral studies. Since flavonoids and alkaloids are known for remarkable anticancer and anthelmintic activities respectively, the researcher is interested to document the research findings on these activities.The present research is aimed to evaluate bioactive principles present in acetone, chloroform and ethyl acetate extracts of Croton Original Research Article Bhargav et al.; JPRI, 33(3): 45-53, 2021; Article no.JPRI.65034 46 scabiosus Bedd. by chemical tests and spectral analysis. It is also aimed to study the anthelmintic and anticancer potential of these extracts.Acetone, chloroform and ethyl acetate extracts of Croton scabiosus Bedd. were prepared by triple maceration technique. GC-MS and HPTLC analysis were performed to identify chemical constituents. Anthelmintic and anticancer potential was assessed for the three extracts. Owing to the positive results of chemical constituents, the researcher made an attempt to evaluate anthelmintic, cytotoxic potential and anticancer activity on breast adenocarcinoma cells (MCF-7) and lung adenocarcinoma cell line (A549). It was pointed to appraise Croton scabiosus Bedd. extracts have considerable Anthelmintic, cytotoxic and anticancer potential.


INTRODUCTION
Herbal medication is a versatile practice generally used by traditional societies. According to the World Health Organization survey of 2000, globally 80% of the world population are practising phytotherapy to cure various diseases. 67% of chemotherapy drugs are from a natural origin [1]. Ayurveda system is also a traditional practice which utilizes plant sources as oral medicine in the form of food, decoctions, powders or external application.
Breast cancers and Lung cancers are the most common cancers in India in terms of prevalence and mortality. According to Globocan 2018 statistics [2] 67,795 new cases and 63,475 deaths occurred due to Lung cancer. In 2018, 1,62,468 new breast cancer cases and 87,090 deaths were reported in India. Chemotherapy with synthetic agents has shown to develop severe side effects. Generally, herbal drugs are used to reduce chemotherapy pertinent side effects and cancer-related effects [3]. The peculiar character of herbal drugs is they can demonstrate better cytotoxic activity only on cancer cells and not on healthy cells by conjugating them with suitable targets. Isoflavones, which were derived from soya bean have accomplished anticancer effects on breast and reduced the probability of developing cancer. Most of the chinese herbal drugs were known to treat Breast cancer, in which most of them have no proven mechanism of action. Medicinal herbs which were popularizedas anti-cancer are being taken as healthy foods or dietary supplements for decades. Pharmacological phytoconstituents isolation, Invivo studies and clinical trials should be conducted for specific targeted applications.  [4]. Croton scabiosus Bedd. aqueous bark decoction is known as a remedy to heal Leucorrhoea and (helminths) worm infections [5] whereas seed extracts were known to be used as an external patch in snake bite [6] to neutralize the poison. But scientific research evidence is not reported for folklore medical practice.
Since secondary metabolites impart several biological activities, this research is intended to identify phytochemical constituents present in Acetone, ethyl acetate and chloroform bark extracts of Croton scabiosus Bedd. by chemical and spectral methods. Hence all three extracts have given the positive result for tannins, alkaloids and saponins as bitter principles; an effort is made to evaluate anthelmintic activity for three extracts. The chloroform and ethyl acetate extracts possess flavonoids and glycosides. These are also tested for cytotoxic potential and a study is made on morphological behaviour against lung adenocarcinoma cell line (A549) and Breast adenocarcinoma cells (MCF-7) to extend the research on evaluation of the anticancer activity. The research outcomes of these methods are specified in the experimental part, illustrate the considerable anthelmintic, cytotoxic and anticancer activities of Croton scabiosus Bedd. (Mention season of collection and scientist who identify the plant).

Preparation of extracts
Triple maceration technique was used to prepare Croton scabiosus Bedd. bark extracts. The bark of Croton scabiosus Bedd.was dried then cut into small pieces later crushed to give a moderately coarse powder. Each 250 g of powder was placed in a round bottom flask. To this 750 ml of the solvent acetone (M1) was added as menstruum to the flask. This was allowed to stand for seven days with occasional shaking. The extracts were concentrated to dryness under reduced pressure using a rotary vacuum evaporator. By following the same procedure extraction was carried out by chloroform (M2) and ethyl acetate (M3) separately as the menstruums. These extracts were subjected to auxiliary studies.

HPTLC
High-performance Thin-Layer Chromatography was performed using silica gel 60F254 (10 cm× 10 cm; 0.25mm layer thickness; Merck) as adsorbent.25mg/ml of each M1, M2 and M3 extracts were filtered separately using a 0.45micron filter paper and these concentrates were exposed to HPTLC (CAMAG, Switzerland). Investigation at three extraordinary fixations 4, 8, 12 μg/ml was spotted independently on a silica gel 60F254 (Merck, Darmstadt, Germany) TLC plate. The TLC plate was developed using Hexane: chloroform: methanol (5:4:1 v/v) as mobile phase in a CAMAG-twin-trough glass chamber previously saturated with mobile phase vapour for 20 min. After developing the plate, it was dried at 65°C for 2 min. It was scanned with Scanner 3 (WinCATS 4 software, CAMAG, Switzerland) at 254 nm and 366 nm [9].

GC-MS
According to sangggil choe, et al the GC-MS study of the components and development were carried out with slight modifications, equipped with a mass-selective detector (MSD) (HP6890 GC and HP7673) auto sampler, operated at 65eV using acquisition scan mode with HP-5MS (GC capillary column, 0.25 mm×0.25 µm×30 m) at 100°C oven temperature held initially for 1 min and then increased gradually to 280°C by 20°C/min and held for 10 min. Injector temperature was maintained at 250°C and Helium was used as the carrier gas at a constant column flow rate of 1.5 ml/min. 2µl of the sample extract was injected by a splitless mode technique. The average of 10 chromatograms were considered to assess the components and registered by Comp Extractor software [10].

Anthelmintic activity
Acetone, chloroform, ethyl acetate Croton scabiosus Bedd. bark extracts were used to evaluate anthelmintic activity on 'Pheretima Posthuma' worms using Albendazole as a reference standard. Three different concentrations (50, 75 and 100mg/ml) of each 20 ml were stacked and six earthworms (same size) were set in it. Both the test and standard solutions were freshly prepared, then earthworms were kept in both test and standard solution to record paralysis time when no waving of worms observed, unless shaken vigorously.
Death time was noted when worms were not moved after replacement of hot water [11].

Cell culture
A-549 and MCF-7 cells were sub-cultured inhouse in CMCR, VIPER Narsapur. The source of the cell line is ATCC. Selected cells were cultured in corning flasks of 75 cm 2 filled with DMEM. DMEM prepared with 10% fetal bovine serum, 1% non-essential amino acids, 1% Penicillin (1000U/mL), 1% Streptomycin (1000μg/mL) and 1% Amphotericin (250 U/mL). Cells were maintained at 37°C with a humidified atmosphere of 5% CO 2 . 0.25% trypsin -EDTA 1mMwas used to passage(30-50) the cells enzymatically in 75 cm 2 corning flasks so as to give 2.2x10 4 cells/cm 2 .Fresh culture medium was supplemented every 2 days. The microbial observance was made periodically to conform 80 % of Cell confluence. After seeding of the cells these were treated with culture medium about 12 hr to prevent cell differentiation. 1:3 to 1:8 was followed for sub cultivation process. The culture medium was restored thrice a week with fresh DMEM [12,13].

Screening of test samples against morphology of A-549 Cells and MCF-7 Cells
A-549 and MCF-7 Cells were treated with 100 and 250 μg/ml concentration of each sample namely M2 and M3 for morphological study.

RESULTS AND DISCUSSION
Phytochemical tests revealed that all three extracts contained various secondary metabolites. The chloroform and ethyl acetate extracts mainly contained higher amounts of terpenoids, Alkaloids, Steroids, flavonoids and carbohydrates, saponins. Inorganic esters like phosphates and sulphates were identified in all three extracts, whereas the acetone extract contained the above constituents at lower amounts as shown in Table 1.

HPTLC
HPTLC analysis was carried on three different Croton scabiosusBedd.extracts to identify specific therapeutically activeprinciples of acetone (M1), chloroform (M2), ethyl acetate (M3) at two different wavelengths (254 and 366 nm) before and after derivatization with a UV detector. Various phytoconstituents identified by HPTLC analysis were represented in Table 2. The major Phytoconstituents identified were Astaxanthin, Colchicine, Reserpine and their R f values were 0.35, 0.29 and 0.48 respectively upon comparison with standard data as depicted in Fig. 1.

GC-MS
Gas chromatography-Mass spectrometry analysis of acetone (M1), chloroform (M2) and ethyl acetate (M3) extracts of Croton scabiosus Bedd. exhibited different peaks and each peak represented principle phyto component of the specific extract. Steroids, fatty acids, terpenes, isoquinoline alkaloids, essential oils etc., were identified as secondary metabolites of Croton scabiosus Bedd. hence they were matched with mass spectra of NIST library. These phytochemicals may accord to the various medicinal activities like anthelmintic, anti-cancer, anti-inflammatory, anti-oxidant etc. The phytoconstituents identified in all the bark extracts of Croton scabiosus Bedd. Along with their retention times were represented in Table 3

Anthelminthic Activity
The results shown in Table 4 reveal that the acetone, chloroform and ethyl acetate extracts of Croton scabiosus Bedd. Showed compelling anthelminthic activity against earthworms in a dose-dependent manner tested in three different concentrations and it is compared with standard Albendazole drug. Among all the three extracts, ethyl acetate extract showed potent anthelmintic activity compared with other extracts.

Results for Morphological Study
Morphological study for the two extracts of Croton scabiosus Bedd. i.e., chloroform and ethyl acetate was performed on A-549 and MCF-7 cell lines. M2 has shown retardation of cellular growth against both A-549 and MCF-7 cells. M3 has shown retardation of cellular growth against A-549 only but not against MCF-7. M2 and M3 extracts at 100 and 250 μg/ml, has shown significant activity against lung adenocarcinoma cell line (A549) and breast adenocarcinoma cells (MCF-7). M2 has shown potent anti-cancer activity against both A549 and MCF-7 cells but M3 has shown anti-cancer activity only against A-549 cells as exhibited in Fig. 3.

Results for Cytotoxic Assay
Cisplatin had shown cytotoxic activity against both A-549 and MCF-7 cancer cell lines at 10 µg/ml concentration as IC 50 . The chloroform and ethyl acetate extracts of Croton scabiosus Bedd. were subjected to cytotoxic assay by MTT assay method against A-549 and MCF-7 cancer cell lines and the research observations were represented in Table 5 and Fig. 4. Upon increase in concentrations from 25µg/ml to 300 µg/ml significant reduction in cytotoxic cells were observed.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.