Swapna R Nayaka¹, Sabari Anand J V², Shaik Mabu Shareef³ and Usha N S^1*

¹Department of Pharmacology, MVJ Medical College and Research Hospital,  Hoskote - 562114, India

²Department of Pharmacology, MGMC, and RI, Mahatma Gandhi Medical College and Research Institute, Pondicherry, India.

³Department of Pharmacology, Kurnool Medical College,  Kurnool - 518002, Andhra Pradesh, India.

Corresponding Author E-mail:  drusha26@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/2069

Abstract

Seaweeds (Marine macroalgae) area large group of marine organisms containing important phytochemical constituents with various biologicalactivities .They are potentially prolific sources of highly bioactivese condary metabolites, which manifest many of the rapeutic effects  like anti-cancer, anti-oxidant, anti-inflammatory and anti-diabetic activities. Seaweeds are used  by many Asian cultures fortraditional medicine preparations. The Caulerpapeltata was collected from Rameshwaram coastal area it was shadedried, madein to powder using standardized procedure to get Caulerpapeltata Methanolic Extract (CPME). The phytochemicals and Gas Chromatography-Mass Spectrometry (GC-MS) analysis was done on prepared CPME for identifying the bioactive compounds Phytochemical in vestigation suggests that the Caulerpapeltata exhibited the presence of phytochemicals like Alkaloids, Carbohydrates, Phytosterols, Saponins and Diterpenes,which may contribute to its biological activities. GC-MS analysis showed 28 variety  of compounds,among which Dibutylphthalate, n-hexadecanoic acid, and1,2–Benzenedic arboxylic acid was found in high percentage. The phytochemical studies and the compounds available in GC–MS showed that the Caulerpapeltata contain important bio active compounds,which may have anti-microbial,anti-fungal and anti-canceractivity. Further research is needed for finding its use in development of new pharmaceutical agent and its safe consumption by human for various health benefits.

Keywords

CPME; Caulerpapeltata; GC-Msanalysis; Phytochemicals

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Copy the following to cite this article: Nayaka S. R, Anand J. V. S, Shareef S. M, Usha N. S. Gas Chromatography – Mass Spectroscopy [ Gc-Ms ] Analysis and Phytochemical Screening for Bioactive Compounds in Caulerpapeltata(Greenalga). Biomed Pharmacol J 2020;13(4).

Copy the following to cite this URL: Nayaka S. R, Anand J. V. S, Shareef S. M, Usha N. S. Gas Chromatography – Mass Spectroscopy [ Gc-Ms ] Analysis and Phytochemical Screening for Bioactive Compounds in Caulerpapeltata(Greenalga). Biomed Pharmacol J 2020;13(4).Available from: https://bit.ly/3m7CMqs

Introduction

Plant -derived compounds are  fascinating  the  world due  to  their  multifaceted therapeutic  use  in  modern medicine. Medicinal plantsare  abundant  bio-resource of drugs  for  traditional system of  medicine,nutraceuticals ,food supplements, folk medicines,pharmaceutical in termediates and chemical entities for developing  synthetic drugs¹_.

Sea weed sor marine macroalgaea replants and ecologically, commercially valua bleliving marine resources that belong to the primitive groups of non-flowering plants without trueroot, stemand leaves i n the division Thallophyta of Plant kingdom.Marine  macro  algae  are  classified into  four groups  based  on  pigments,  stored  food  materials,  morphological  and  anatomical  characters  into Chlorophyta (Green seaweeds), Phaeophyta (Brown seaweeds), Rhodophyta (Redseaweeds) and Cyanophyta (Bluegreenalgae)². Seaweeds obtained  from coastal  region  are  the  potential marine  resource  for  many  biochemical compounds.Pharmaceutical  importance  of  seaweeds  globally  has  led  to  ample  research to extract  out  bio active  compounds from algae. Marine habitat is an abundant  resource of functional materials  such  as  poly unsaturated fatty acids, poly saccha rides,essential minerals and vitamins, antioxidants, enzymes and bio active peptides³.

The nutritional value of a food depends on its chemical composition such as carbohydrates, proteins, lipids, sugars and also the minerals present in them. Sea weeds are the rich source of bio active compounds like carotenoids, dietary fiber, protein,essential fatty acids, vitamins and mineral⁴ and they contain biologically active substances such  as lipids, proteins, poly sacch a rides and poly phenol⁵.   The bio chemical composition of sea weeds differs, which are affected by in flow of land sources, geographic areas as on of they ear and temperature of water⁶ 

Recently, the  importance of sea weeds as a source of novel bio active sub stances is growing rapidly and researchers have revealed that their  compound sex hibit various biological activities due to the secondary meta bolites⁷. The  bio active  compound sex tracted  from different  Marine  algae  have antioxidant ,antiviral ,anti fungal and anti microbial activities⁸.

Caulerpapeltata is marine green algae with small fleshy‘ umbrellas’   is sometimes seen on our southern shores growing on coral rubble, near reefs. The species shave several benefits for which it is consumed as food  and  also  as medicine for its anti fungal properties & its potential to reduce blood pressure.Although,there are no  comprehensive studies regarding the active components in this seaweed. Hence,  the current research work has focused on the phyto chemical profiling of Caulerpapeltata and the active biochemical compounds were further identified using gas chromato graphic mass spectrometry analysis.

Materials and Methods

Collection  of Seaweed

Caulerpapeltata was collected from the vedalaicoastal area of Rameshwaram, TamilNadu, India and Marine  Biologist  helped in identifying the sample. The herbarium of the Caulerpapeltata was prepared and stored.

Preparation of seaweed Material

After collection of the seaweed it was washed many times to remove epiphytes, sand particles, other species parts, etc. The collected sample was soaked in distilled water twice. After thorough washing, seaweed sample was shade dried, till it becomes moisture free.

Preparation of extracts 

A dried sample of seaweed was ground into coarse powder in a mechanical grinder. The sample was subjected to maceration at 24- 25°C in95%  methanol for about 72 hours. The methanolic extract was derived after the process of distillation, evaporation, and drying it under reduced pressure⁹.

Gas Chromatography-Mass Spectroscopy analysis

GC-MS analysis performed by using a Shimadzu QPQP-2010 Plus with Thermal Desorption System TD20.

Derivatization of plant extracts for GC-MS: In the ratio of 1:4, water and ethyl  acetate was added to the separating funnel containing concentrated sample. Small amount of concentrated sample was taken in a separating funnel and shaken by adding in 1:4 ratio. The upper layer was collected and concentrated to 1.5 mlin therotary evaporator, which is taken in funnel to add 100µl N,O-Bis(trimethylsilyl) tri fluoro acetamide and trimethyl chlorosilane(BSTFA+TMCS) & 20µl of Pyridine and heat it at 60°Cf or half an hour. To this sample Aceto nitrile was added & filtered in to aconical flask to which 50µl BSTFA+TMCS was added and heated again at 60°C  in a water bath for 30 minutes. The CPME was filtered usin g0. 45µ membrane filter to avial ¹⁰.

Identi fication of phyto constituents

The inter pretation of Mass-Spectrum  of  GC-MS of the unknown compound was compared with the known components using database stored in National Institute Standard and Technology (NIST).

Qualitative analysis of phyto chemical substance The sample was subjected to phyto chemical analysis for detection of alkaloids, carbohydrates,  glycosides,  saponins,  phytosterols,  phenols,  tannins,  flavonoids,  diterpenes,  proteins  &  amino  acid using the standard qualitative procedures ^{11 -13}.

Results and Discussion:

The extract wastested for various phyto chemicals & constituents like alkaloids, saponins, phytosterols, diterpenes & carbohydrates were recorded in C.peltata  are shown in the Table No.1.

Table 1: Phyto chemicals presenting C.peltata methanolic extract.

Phytochemicals	Chemicalanalysis&results
Alkaloids	Wagner’stest++, Hager’stest++ Dragendroff’stest++
Saponins	Frothtest++, Foamtest++
Phytosterols	LiebermannBurchard’stest++
Diterpenes	Copperacetatetest++
Carbohydrates	Molisch’stest++, Benedict’stest++ Fehling’stest++

Alkaloids in seaweeds have anti microbial, anti diarrheal & anthelminticaction³. In present study Caulerpapeltata methanolic extract contains alkaloids, which suggests that it can be used as medicinal drug.

Saponinsare integral component of Chinese medicine because it has discrete properties responsible for most of the biological actions.  Hence,  seaweeds use dinhyper cholesterolemia, hyperglycemia,  to reduce weight & as antioxidant, anticancer, anti-inflammatory agents¹⁴. Our study confirms presence of Saponins in methanol extract of C.peltata, suggesting that its benefit in suppressing infections& inflammation caused due to bacteria  &  fungi.

The extract of C. peltata, can be used as antimicrobial  & anti diarrhoeal agents as it consists of both phytosterols &  diterpenes³. As Steroids have been reported to have anti bacterial properties ¹⁵.

Bio active compound sextensively researched in carbohydrate are sulfated polysaccha rides which possess good antibacterial, anti-viral activity ¹⁶. In our study C.peltata extract tested positive for carbohydrates suggesting that it can be used in medical field as anti bacterial & anti viral agents.The algaldietary fiber consists soluble poly saccharides having salient action s such as antioxidant, anticoagulant, anti mutagenic and anti tumour effect and have an key role in the modification of lipid metabolism in human body ¹⁷.

The results observed  by Raja sulochana  Petal, Venkatesh  R. Petal, Jayasree  NB Petal, on phyto chemical compounds ^18-20 help our study on the usefulness of marine algae in traditional medicine and added anoteonthephy to chemical history.Due the presence of phyto constituents in the seaweeds, they are beneficial in obtaining drugs for  treating various ailments in humans. The sephyto constituents possess anti bacterial ²¹, antiviral ²²,anti fungal ²³,anticoagulant,anti-tumor ²⁴ and anti-inflam matory activities ²⁵.

GC-MS analysis was conducted in crude extract of Caulerpapeltata.The current study 28 different biochemical compounds have been identified from methanolic extract of the algae Caulerpapeltata.The compounds exhibited a wide range in their nature. Largest peak area was observed for the compound Dibutyl phthalate & other phyto chemicals liken-hexadecanoic acid&1,2-Benzene dicarboxylic acid also have alargearea.The compound with highest molecular weight was Hexacosyl penta fluoropro pionate (Figure No.1 & table No.2).Major phyto chemical’s with their chemical structures mentioned in table 3.

The most abundant phyto compounds,Dibutylphth alateactsas anti metabolic agent ²⁶,n-hexadecanoic acid has various bio logical activities like antioxidant, hypolipidaemic, nematocide, lubricant  & haemolytic  inhibitor ²⁷,where as 1,2-Benzene dicarboxylic acid has anti microbial & anti fouling activity ²⁸.Hence C.peltata should be extensively studieds otat it can be used in pharmaceutical industry for their medicinal value.

Figure 1: Chromatogram analysis of C.peltata Click here to View figure

 

Table 2: Phyto components in methanolic extract of Caulerpapeltata.

Sl.No.	ReactionTime	Area%	Mol.weight	Compoundname	MolecularFormula
1	7.163	1.33	115	Acetamide,N,N-diethyl-	C6H13NO
2	7.382	1.57	87	Acetamide,N-Ethyl-	C4H9NO
3	13.563	0.58	204	Caryophyllene	C15H24
4	14.543	0.67	206	Phenol,3,5-bis(1,1dimethylethyl)-	C14H22O
5	15.533	2.52	222	DiethylPhthalate	C12H14O4
6	16.482	0.48	226	8-Pentadecanone	C15H30O
7	17.444	0.78	228	Tetradecanoicacid	C14H28O2
8	17.819	1.23	238	1-Heptadecene	C17H34
9	18.278	0.91	278	Neophytadiene	C20H38
10	18.542	0.62	278	1,2-benzenedicarboxylicacid,bis(2-methylp	C16H22O4
11	18.666	1.33	226	8-Pentadecanone	C15H30O
12	19.175	2.73	270	Hexadecanoic acid, methylester	C17H34O2
13	19.519	42.02	278	Dibutylphthalate	C16H22O4
14	19.552	17.24	256	n-Hexadecanoic acid	C16H32O2
15	19.865	1.65	266	1-Nonadecene	C19H38
16	19.986	0.84	340	Eicosylacetate	C22H44O2
17	20.646	0.58	282	10-Nonadecanone	C19H38O
18	20.813	1.33	268	Oxirane,hexadecyl-	C18H36O
19	20.872	0.41	296	MethylDihydromalValate	C19H36O2
20	21.225	0.96	282	Heptadecene-(8)-CarbonicAcid-(1)	C18H34O2
21	21.438	3.36	284	Octadecanoicacid	C18H34O2
22	21.733	1.49	410	Octacosanol	C28H58O
23	21.843	1.16	340	Eicosylacetate	C22H44O2
24	23.446	1.00	354	n-Tetracosanol-1	C24H50O
25	24.562	10.22	390	1,2-Benzenedicarboxylicacid	C24H38O4
26	25.030	0.51	396	1-Heptacosanol	C27H56O
27	27.861	0.92	528	Hexacosylpentafluoropropionate	C29H53F5O2
28	31.520	1.55	386	Cholesterol	C27H460

 

Table 3: Major phytochemical compounds in the methanolic extract of Caulerpapeltata & their chemical structure. Click here to View table

Conclusion

The current study confirmed  28 distinct phytochemical substances present in  the extract of  Caulerpapeltata  which could be the bio active constituents.  ThealgaC.peltata are valuable reservoir of bio active compounds of medicinal interest which needs detailed studies so that it may beusedindrug for mulation by the pharmaceutical industries.

Acknowledgment

We acknowledge Dr S Bragdeeswaran, Associate  Professor CAS in Marine biology,  Annamalai University, who really gave invaluable support  & contribution in the study.

Conflict of Interest

Authors declare there is no conflict of interest

Funding Source

There is no funding source

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