HR-LCMS and In vitro cytotoxicity analysis of Alseodaphne semecarpifolia stem bark and leaf methanol extracts

 

Ganadhal Puttaramaiah Chethankumara1, Venkatarangaiah Krishna2, Kakanahalli Nagaraj1*

1Department of PG Studies and Research in Applied Zoology,

Kuvempu University, Shivamogga, Karnataka, 577 451.

2Department of PG Studies and Research in Biotechnology, Kuvempu University, Shivamogga, Karnataka, 577 451

*Corresponding Author E-mail: knagarajv@gmail.com

 

ABSTRACT:

A. semecarpifolia (Lauraceae) has been employed by Indian traditional healers to treat various human cancers. The cytotoxic property of A. semecarpifolia was evaluated by extracting secondary metabolites from stem bark and leaves. The stem bark methanol extract (SBME) and leaf methanol extract (LME) were subjected to HR-LCMS analysis, followed by evaluating their cytotoxic potential against MCF-7, SCC-9, HeLa, A-549 and L6 cells by MTT assay. The phytochemical screening of SBME and LME revealed the presence of alkaloids, flavonoids, terpenoids, steroids, saponins, phenolics and glycosides. HR-LCMS analysis of SBME and LME revealed the presence of 31 and 28 active compounds respectively. Among them Cucurbitacin A and Gambogic acid are the two compounds identified with anticancer properties. SBME showed potent cytotoxicity on MCF-7 and SCC-9 cells, whereas LME showed significant cytotoxicity only on MCF-7 cells. Vinblastine sulphate was used as a reference standard, even though Vinblastine is an efficient anticancer drug it affected the viability of normal cells. In comparison with Vinblastine, both SBME and LME showed very less toxicity on normal cells. Hence, the present study suggested that stem bark and leaves of A. semecarpifolia are the possible chemotherapeutic agents having potential cytotoxic activity against MCF-7 and SCC-9 cells.

 

KEYWORDS: Alseodaphne semecarpifolia, Cell lines, Cytotoxicity, HR-LCMS, Phytochemicals.

 

 


INTRODUCTION:

Medicinal plants are the reservoirs of novel chemical compounds and many of them have been screened for their pharmacological activities1. The global demand for herbal medicines is steadily increasing and numerous drugs have entered into the market through exploration of traditional medicines2. The bioactive phytochemicals are frequently used as substitute for modern medicines. In traditional medicinal system, many of the plants are being used as an alternative approach to minimise the irreversible effects of modern drugs on the immune system3,4. The potential of some herbal formulations are also recognized in the prevention and treatment of cancer5.

 

Cancer affects normal metabolism in the body, regardless of various developments in diagnosis, treatment and prevention of the disease6. Even though radiation therapy and chemotherapy are effective treatments for cancer, they are accompanied by various unbearable side effects causing harmful effect on healthy cells, thus new approaches are required to solve these problems7. The interest in development of natural anticancer drugs is growing with time and natural products are accepted for exploration of new anticancer agents due to their high toxicity on cancer cells and less toxicity on normal cells8. Nevertheless, plant derived compounds are safer and less toxic over conventional treatment and these plant compounds act specifically on cancer cells without affecting the normal cells9. Hence, the anticancer potential of natural drug molecules has been studied in several experimental models, in order to validate their efficacy10.

 

A. semecarpifolia is an evergreen tree of the lauraceous family11. The ethno medicinal survey has revealed that the traditional medicinal practitioners in Central Western Ghats of Karnataka, India employed stem bark and leaves of A. semecarpifolia to treat various human cancers. Even though the plant is employed in traditional practices over the years, it is lacking support from sufficient scientific approaches. Hence, the present investigation was initiated to evaluate its cytotoxic potential against human cancer cells.

 

MATERIALS AND METHODS:

Plant material collection and extraction of secondary metabolites:

The stem bark and leaves of A. semecarpifolia were collected from Karigudda, Central Western Ghats region of Karnataka, India. The plant was identified and authenticated by Dr. Y. L. Krishnamurthy, Taxonomist, Department of Applied Botany, Kuvempu University, Shivamogga, Karnataka, India. The voucher specimen (KUBPHS78) is deposited in the herbarium of DBT-Builder project, Kuvempu University. The collected samples were subjected to sequential Soxhlet extraction using petroleum ether, chloroform and methanol. The obtained extracts were concentrated using rotary evaporator and desiccated until further analysis.

 

Phytochemical screening:

The phytochemical analysis was carried out qualitatively and quantitatively using the standard procedures12-24. Based on the percentage yield of the extract and presence of phytochemical constituents, stem bark methanol extract (SBME) and leaf methanol extract (LME) were selected for HR-LCMS and cytotoxicity analysis.

 

HR-LCMS analysis of stem bark and leaf methanol extracts:

The phytochemical constituents present in SBME and LME were identified by comparing their retention time (RT) and mass with stored metlin library available in Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology (IIT), Mumbai, India using High Resolution Liquid Chromatograph Mass Spectrometer (G6550A system, Agilent technologies, USA) equipped with electrospray ionization (ESI). The data was acquired by 5µl sample injection for 30 minutes by coupling ESI with double mass spectrometry units (30 minutes + ESI 10032014_MSMS.m.). The gas temperature of 250°C was used for the analysis and the phytochemical constituents were identified by theoretical mass of protonated compound.

 

Screening for cytotoxic activity:

The cell lines were cultured by using Dulbecco’s Modified Eagle Medium (DMEM) by supplementing with streptomycin, 10% inactivated Fetal Bovine Serum (FBS) and penicillin in a humidified atmosphere of 5% CO2 at 37°C. The cells were seeded 50,000 cells/well in a 96 well plate. They were allowed to form a monolayer under regular growth conditions and these cells were harvested for cytotoxicity studies. The cytotoxic effect of SBME and LME on MCF-7 (Breast cancer), SCC-9 (Oral cancer), A-549 (Lung cancer), HeLa (Cervical cancer) and L6 (Normal rat myoblast cells) cells was determined by MTT assay25. 100µl of the diluted cell suspension was loaded onto the respective wells in 96 well plate and incubated for 24 hours in CO2 incubator at 37°C with 5% CO2, 95% air and 100% relative humidity. After incubation, the supernatant was carefully flicked off when the partial monolayer was observed, followed by washing with DMEM. Later the cells were treated with different concentrations of test samples (10, 20, 40, 80, 160 and 320µg/ml) and incubated for 24 hours in CO2 incubator with same incubation conditions. After incubation, the test solutions in the wells were exchanged with 100µl (0.5mg/ml) of MTT solution and again incubated for 4 hours in CO2 incubator. The supernatant was removed and 100µl of DMSO was added and the plate was gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader (SpectraMax i3) at a wavelength of 590nm. The IC50 values were obtained from the dose-response curves and the percentage growth inhibition was calculated.

 

Statistical analysis:

The statistical analysis was performed using GraphPad Prism Software v 5.01 (GraphPad Software Inc., San Diego, CA). The data is presented as mean±SEM of three replicates and it is statistically analyzed using two way analysis of variance (ANOVA) followed by Bonferroni post-test. The ‘p’ value less than 0.001 was deemed to be statistically significant.

 

RESULTS:

Extraction yield:

The soxhlet extraction yield of stem bark petroleum ether extract (SBPEE), stem bark chloroform extract (SBCE) and stem bark methanol extract (SBME) was 4.5g (0.45%), 12g (1.2%) and 62g (6.2%) respectively. The extraction yield of leaf petroleum ether extract (LPEE), leaf chloroform extract (LCE) and leaf methanol extract (LME) was 6.8g (0.68%), 14.5g (1.45%) and 80g (8.0%) respectively.

 

Qualitative phytochemical analysis:

The qualitative phytochemical analysis of stem bark extracts showed that SBPEE contains only tannins and steroids. Alkaloids, flavonoids, tannins, steroids and glycosides are the phytochemicals present in SBCE, whereas, SBME contains alkaloids, flavonoids, glycosides, phenols, saponins and terpenoids. It is noteworthy that phenols, saponins and terpenoids are present only in SBME, alkaloids and flavonoids are present only in SBCE and SBME. Tannins and sterols are present only in SBPEE and SBCE.


Table 1: Qualitative phytochemical analysis of A. semecarpifolia stem bark and leaf extracts.

Sl NO.

Phytochemical Constituents

Stem Bark Extracts

Leaf Extracts

SBPEE

SBCE

SBME

LPEE

LCE

LME

01

Alkaloids

-

+

+

-

+

+

02

Flavonoids

-

+

+

+

-

+

03

Tannins

+

+

-

+

+

-

04

Steroids

+

+

-

+

-

+

05

Glycosides

-

+

+

+

-

+

06

Phenols

-

-

+

-

+

+

07

Saponins

-

-

+

-

-

+

08

Terpenoids

-

-

+

+

-

+

+ Present – Absent

 

Table 2: Quantitative phytochemical analysis of A. semecarpifolia stem bark and leaf extracts.

Sl

No.

Phytochemical constituents

Stem Bark Extracts

Leaf Extracts

SBPEE (µg/mg)

SBCE (µg/mg)

SBME (µg/mg)

LPEE (µg/mg)

LCE (µg/mg)

LME (µg/mg)

01

Total Alkaloids

-

69.24±7.17

113.45±3.28

-

49.05±2.48

148.86±5.46

02

Total Flavonoids

-

75.66±19.07

134.16±7.76

32.16±1.46

-

104.73±4.89

03

Total Tannins

55.34±9.37

22.11±10.31

-

25.33±11.19

21.48±5.03

-

04

Total Steroids

43.66±14.70

17.26±4.67

-

13.41±6.73

-

30.18±12.40

05

Total Glycosides

-

30.48±6.02

9.32±2.41

8.36±1.31

-

12.96±9.20

06

Total Phenolics

-

-

71.76±6.26

-

35.54±6.97

98.64±6.76

07

Total Saponins

-

-

62.46±11.06

-

-

87.23±3.36

08

Total Terpenoids

-

-

26.16±8.27

8.26±4.17

-

21.36±8.09

 


Similarly, qualitative phytochemical analysis of leaf extracts revealed that LPEE contains flavonoids, tannins, steroids, glycosides and terpenoids. LCE showed the presence of only alkaloids, tannins and phenols. Whereas, LME revealed the presence of alkaloids, flavonoids, steroids, glycosides, phenols, saponins and terpenoids. The results of the qualitative phytochemical analysis are presented in Table 1.

 

Quantitative phytochemical analysis:

The quantitative phytochemical analysis showed the presence of secondary metabolites in different concentrations of dry weight of the extracts. SBPEE revealed the presence of only tannins and steroids with 55.34±9.37µg/mg and 43.66±14.70µg/mg concentrations respectively. SBCE is rich in flavonoids (75.66±19.07µg/mg) followed by alkaloids (69.24±7.17µg/mg). Likewise, SBME is rich in flavonoids (134.16±7.76µg/mg) and alkaloids (113.45±3.28µg/mg) followed by phenolics (71.76±6.26µg/mg) and saponins (62.46±11.06µg/mg).

 

LPEE is rich in flavonoids (32.16±1.46µg/mg), whereas, LCE is rich in alkaloids (49.05±2.48µg/mg) and phenolics (35.54±6.97µg/mg). Furthermore, LME is very rich in alkaloids (148.86±5.46µg/mg) and flavonoids (104.73±4.89µg/mg) followed by phenolics (98.64±6.76µg/mg) and saponins (87.23±3.36µg/mg). The results of quantitative phytochemical analysis are presented in Table 2.

 

HR-LCMS analysis of stem bark and leaf methanol extracts:

HR-LCMS analysis of SBME and LME has revealed the presence of 31 and 28 phytochemicals respectively (Table 3 and 4; Figure 1 and 2). The major compounds identified in SBME are Nadolol, Carboxyterbinafine, Cucurbitacin A, Carboxyprimaquine, Amiprilose, (S)-Reticuline, Methadone, Esmolol, D-Urobilinogen, Gambogic acid, Neomycin C, Lobeline and Erythroxanthin Sulfate. The major compounds identified in LME are Acarbose, Ondansetron, Norfloxacin, Methylergonovine, Norcisapride, Pipenzolate, Methysergide, Dextromoramide M4, Desmethylgliquidone and Ofloxacin. Among these compounds, Cucurbitacin A and Gambogic acid are known for their anticancer properties26-30.


 

Table 3: Phytochemical constituents of A. semecarpifolia SBME identified by HR-LCMS analysis.

Sl No.

Compound Name

Retention Time (min)

Mass

Formula

01

Nadolol

1.044

309.1939

C17H27NO4

02

Carboxyterbinafine

1.057

321.1736

C21H23NO2

03

Ser Lys Asn

1.081

347.1779

C13H25N5O6

04

Cucurbitacin A

1.084

574.305

C32H46O9

05

Carboxyprimaquine

1.115

274.1344

C15H18N2O3

06

Amiprilose

1.413

305.1872

C14H27NO6

07

Norpropoxyphene

1.613

325.2042

C21H27NO2

08

Gly Asn

1.958

189.0742

C6H11N3O4

09

Thr Leu Asp

2.159

347.1714

C14H25N3O7

10

(S)-Reticuline

2.556

329.1621

C19H23NO4

11

Met Val Pro

2.561

345.1715

C15H27N3O4S

12

Methadone

3.576

309.2071

C21H27NO

13

Terbinafine metabolite

3.949

313.1673

C19H23NO3

14

Esmolol

4.092

295.179

C16H25NO4

15

Gln Gly Ala

4.237

274.1292

C10H18N4O5

16

Gln Ala Asn

4.279

331.1483

C12H21N5O6

17

Piperidolate

4.339

323.1893

C21H25NO2

18

Ala Thr Phe

4.522

337.165

C16H23N3O5

19

1-Propanamine, N,Ndimethyl-3-(5-oxidodibenzo[b,e]thiepin-11(6H)-ylidene)-, (E)- (9CI)

4.671

311.1331

C19H21NOS

20

D-Urobilinogen

4.75

590.3164

C33H42N4O6

21

Phe Gly Val

4.767

321.1685

C16H23N3O4

22

Gambogic acid

4.82

628.3059

C38H44O8

23

Desmethylergometrine

4.823

311.1633

C18H21N3O2

24

Harderoporphyrinogen

4.909

614.2939

C35H42N4O6

25

Pro Phe Thr

4.979

363.1775

C18H25N3O5

26

Neomycin C

5.156

614.3146

C23H46N6O13

27

Tribenzylamine noxide

5.203

303.1652

C21H21NO

28

N-(5Z,8Z,11Z,14Zeicosatetrayoyl)- ethanolamine

5.299

339.2194

C22H29NO2

29

Lobeline

5.302

337.2034

C22H27NO2

30

Erythroxanthin sulfate

7.938

678.3591

C40H54O7S

31

GPSer(10:0/10:0)

9.052

567.3108

C26H50NO10P

 

Table 4: Phytochemical constituents of A. semecarpifolia LME identified by HR-LCMS analysis.

Sl No.

Compound Name

Retention Time (min)

Mass

Formula

01

Pro Pro Leu

3.951

325.2019

C16H27N3O4

02

Met Val Pro

4.102

345.1714

C15H27N3O4S

03

Pro Pro Phe

5.061

359.1836

C19H25N3O4

04

L-4-Hydroxy-3-methoxy-amethylphenylalanine

5.624

225.102

C11H15NO4

05

N,N-dimethylhistidine

5.647

183.0977

C8H13N3O2

06

Cys Leu Glu

6.366

363.1449

C14H25N3O6S

07

Acarbose (component 1)

6.671

303.1309

C13H21NO7

08

Ondansetron

6.679

293.1517

C18H19N3O

09

Zolpidem Metabolite X-1

6.788

323.1648

C19H21N3O2

10

Norfloxacin

6.895

319.1336

C16H18FN3O3

11

Methylergonovine

7.151

339.1957

C20H25N3O2

12

Mebendazole metabolite (Carbamic acid, [5-

(hydroxyphenylmethyl) 1Hbenzimidazol- 2-yl]- methyl ester

7.291

297.1107

C16H15N3O3

13

Norcisapride

7.424

313.1161

C14H20ClN3O3

14

Met Gly His

7.478

343.1314

C13H21N5O4S

15

Pipenzolate

7.782

353.2012

C22H27NO3

16

Methysergide

8.014

353.2115

C21H27N3O2

17

Cys His Ala

8.099

329.1148

C12H19N5O4S

18

Ofloxacin-N-oxide

8.107

377.1393

C18H20FN3O5

19

Dextromoramide M4

8.297

339.182

C21H25NO3

20

Desmethylgliquidone

8.42

513.1961

C26H31N3O6S

21

Ofloxacin

8.537

361.1441

C18H20FN3O4

22

5-[[4-(4-fluorophenyl)-3-piperidinyl]methoxy]-2-

methoxy-, (3S-trans)-Phenol

8.542

331.1656

C19H22FNO3

23

Asn Phe Trp

8.678

465.1981

C24H27N5O5

24

Cys Leu Ala

8.775

305.144

C12H23N3O4S

25

6alpha-Glucuronosylhyodeoxycholate

8.987

567.3038

C30H47O10

26

(20S,24R)-20-fluoro-1alpha,24-dihydroxy-26,27-

cyclovitamin D3 / (20S,24R)-20-fluoro-1alpha,24-dihydr

10.311

432.3079

C27H41FO3

27

4- Hydroxyphenylretinamide

14.221

391.2492

C26H33NO2

28

Harderoporphyrinogen

27.157

614.3128

C35H42N4O6

 

Fig. 1: HR-LCMS Chromatogram of A. semecarpifolia SBME.

 

Fig. 2: HR-LCMS Chromatogram of A. semecarpifolia LME.

 


Cytotoxic effects of stem bark and leaf methanol extracts:

The cytotoxic activity of SBME and LME was observed in a dose dependent manner and it is expressed as inhibitory concentration 50 (IC50). SBME showed significant cytotoxic activity on MCF-7 (54.45±1.35µg/ml) and SCC-9 (64.87±2.34µg/ml) cells. Whereas, it showed moderate activity on A-549 (151.34±1.74µg/ml) cells and it failed to exert significant effect on HeLa cells. LME exerted significant cytotoxic activity on MCF-7 (58.67±2.31µg/ml) cells. Whereas, it showed moderate activity on HeLa (130.98±2.45µg/ml) and SCC-9 (116.76±2.78µg/ml) cells and it failed to show significant effect on A-549 cells. The standard drug Vinblastine exhibited significant effect on all the four cancer cells. The IC50 of Vinblastine on MCF-7, SCC-9, A-549 and HeLa cells was 24.03±2.12µg/ml, 26.88±2.56µg/ml, 22.67±3.67µg/ml and 21.73±2.67µg/ml respectively. Vinblastine also affected the viability of L6 (88.52±3.56µg/ml) cells, but the IC50 of SBME and LME on L6 cells was unable to determine due to their lesser toxicity even at the higher concentrations tested (Figure 3).


 

Fig. 3: Cytotoxic effects of A. semecarpifolia SBME, LME and Vinblastine against HeLa, SCC-9, A-549, MCF-7 and L6 cells.

 


DISCUSSION:

The employment of the natural products as traditional medicines is growing with time and many of the drugs employed today are derived from exploring traditional medicinal claims31. The attention has been increased towards the utilization of secondary metabolites for the prevention and treatment of various human diseases32. Plant secondary metabolites function in conjunction with one another or they may act alone in order to bring desired pharmacological effects33. The discovery of herbal drugs with minimal adverse side effects is affected by lack of proper standardization. Therefore it is essential to use standardized and advanced scientific screening methods to validate and maintain herbal drug standards34.

 

The main target of in vitro cytotoxicity studies is to measure cell death and growth impairment, and they are designed specially to evaluate the efficacy of the target drug to kill the cancer cells35. For the study of proliferation and cancer progression the use of the appropriate in vitro model in cancer research is crucial36. In the development of new therapeutic drugs for cancer treatment many cell lines have been widely used and proved to be appropriate tool in biological research . They are the excellent model for the study of biological mechanisms involved in cancer37.

 

In the present study, Cucurbitacin A and Gambogic acid identified by HR-LCMS analysis are the two compounds found to have anticancer properties. Cucurbitacin A is a biochemical compound belongs to terpenoid group, it is known for its cytotoxic properties and currently under study to screen its potential anticancer properties26. Gambogic acid is a potential natural compound having a promising role in colorectal cancer therapy and found to inhibit the cancer cell growth by inducing apoptosis27,29,30. It is a xanthonoid derived from Garcinia hanburyi Hook f. used for tumor suppression28,29. Since the last decade, Gambogic acid has been developed as a potential anticancer agent that demonstrate its antimetastatic, antiangiogenic and antitumor activities against certain cancer cells29.

 

The purpose of using anticancer drugs is to inhibit the cancer cell proliferation without affecting the normal cells. From the present study it is evident that SBME and LME affected the viability of cancer cells but not normal cells. Hence, the present study supports the traditional medicinal claims of stem bark and leaves of A. semecarpifolia as in vitro cytotoxic agents against human breast cancer cells and oral cancer cells. Further research is under progress to isolate and characterize bioactive compounds which might lead for the development of new therapeutic compounds to treat human cancers with very less or no toxicity on normal cells.

 

ACKNOWLEDGEMENT:

This work was financially supported by DBT, New Delhi, India through DBT-BUILDER project (Grant No. BT/PR9128/INF/22/190/2013, Dated: 30/06/2015). The authors are thankful to Skanda Lifesciences Pvt Ltd, Bangalore, Karnataka, India and department of Applied Zoology, Kuvempu University, Karnataka, India for providing the laboratory facility to carry out this research. We are also grateful to Sophisticated Analytical Instrument Facility (SAIF), Indian Institue of Technology (IIT), Bombay, for providing analytical facility.

 

CONFLICT OF INTEREST:

The authors declare that, there are no conflicts of interest for this study.

 

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Received on 06.10.2020            Modified on 19.12.2020

Accepted on 24.02.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(1):250-256.

DOI: 10.52711/0974-360X.2022.00041