Identification of Components In Boerhavia Diffusa Plant Extract And Assessment Of Its Biological Activity By Gas Chromatography-Mass spectrometry


Department of Biochemistry, Vels Institute of Science, Technology and Advanced Studies, Pallavaram, Chennai, Tamilnadu-600117, India, 9941709668

Abstract

Boerhavia diffusa is a medicinal plant used for various therapeutic benefits ant the plant can be found in tropics and subtropics regions. It has the pharmacological activities which include antifibrinolytic, immunosuppressive, diuretic, hepatoprotective, antidiabetic, anti-inflammatory, anticancer, immunomodulatory, antilympho proliferative, analgesic properties and also for the treatment of pulmonary tuberculosis. This study deals with the determination of possible phytocompounds present in the hydroethanolic extract of the plant Boerhavia diffusa using Gas chromatography- Mass Spectroscopy technique. Using GC-MS technique the phytocompounds present in the hydroethanolic extract of the plant Boerhavia diffusa was investigated and the mass spectra of the compounds found in the extract were matched with National Institute of Standards and Technology library. GC-MS Analysis of Boerhavia diffusa extract reveals that the existence of phytocompounds which includes 1, 2-Benzenedicarboxylic acid, diethyl ester, Hexadecanoic Acid, 1,2-Benzenedicarboxylic acid, dibutyl ester and 1,2-Benzenedicarboxylic Acid, Dioctyl Ester. This research paper helps to identify the phytocompounds which are present in the extract of Boerhavia diffusa and also prove the biological activities of the phytocompounds present. Furthermore, studies were made in-depth to find the mode of action of the phytocompounds.

Keywords

Boerhavia diffusa, hydroethanolic extract, Gas chromatography- Mass Spectroscopy technique, phytocompounds, biological activity

Introduction

Boerhavia diffusa is popularly known as Punarnava (Indian Medicine), which means rejuvenates or renews the body (Rajpoot & Mishra, 2011). The plants Boerhavia diffusa are seen in both tropical and subtropical regions all over the world (Struwig & Siebert, 2013). It is a medicinal herb in India and the roots used for the treatment of anasarca, ascites, inflammation, piles, and jaundice. They act as a diuretic and laxative agent (Mahesh, Kumar, Mk, & Devkar, 2012). Boerhavia diffusa has thick, deep penetrating and single taproot as shown in Figure 1. The root is stout and fusiform and they have creepy stem arise from the root, swollen at the nodes. They have slightly rounded leaves, as shown in Figure 2. Deep pink color flowers are seen in the plant Boerhavia diffusa (Agrawal, Das, & Pandey, 2011).

Most important bioactive Phytochemical constituents present in the plant Boerhavia diffusa are the glycosides, alkaloids, flavonoids, tannins, steroids, terpenoids, essential oils and phenolic compounds. The number of flavonoids and phenols are higher than the other phytochemicals and they are responsible for many pharmacological activities. Tissues are protected against oxygen free radicals due to its antioxidant property of flavonoids. Thus they prevent cancer, atherosclerosis and chronic inflammation (Beegum, Beevy, & Sugunan, 2014).

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Figure 1: The whole plant of Boerhavia diffusa

Scientific Classification

Botanical name : Boerhavia diffusa

Kingdom: Plantae

Order: Caryophyllales

Family: Nyctaginaceae

Genus: Boerhavia

Species: diffusa

In India six species were found, they are B.diffusa, B.erecta, B.rependa, B.chinensis, B.repens and B.rubicunda and commonly called as Mookarattai.

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Figure 2: The Leaves of Boerhavia diffusa

Benefits of Mookarattai

Boerhavia diffusa used to cure a renal disorder, which may decrease the urea levels in the body. They plant able to cure arthritis and reduce pain and inflammation. They enhance erection, quality and quantity of semen. It increases appetite, reduces abdominal pain and relieves constipation. It acts as a diuretic which may cure kidney stone and also acts as a laxative and detoxifier, which may cure hepatitis. They regenerate the whole body (Kanagavalli, Sadiq, & Lakshmipriya, 2018). Boerhavia diffusa can also cure stomach problems, fever, diarrhoea, dysentery and skin disease. All the parts of the plant have its unique medicinal property (Panda, 2014).

Materials and Methods

Collection of the Plant sample

The samples of plant Boerhavia diffusa Linn was collected from their characteristic natural surroundings and from the market. The plant Boerhavia diffusa was washed in tap water, air-dried and made to a fine powder by a homogenizer. The dry powder acquired was maintained at room temperature in airtight containers.

Preparation of Plant Extract

5 g of dry powdered plant material was extracted with 100 ml of 70% of ethanol for maceration periods (24 hr). At 150 rpm the extraction was agitated and carried out at room temperature. Through Whatmann No. 1 filter paper, the soaked powder-solvent mixtures were filtered after the maceration periods. A Part of the filtrate was used for phytochemical analysis and the remaining solvent evaporated to dry for in vitro antioxidant studies.

Table 1: Identification of compounds in a sample using GC-MS

Peak #

R.Time

Area %

Molecular formula

Molecular weight

Name of the compound

1

7.276

0.63

C14 H30

198

Tetradecane

2

7.541

3.35

C10H10O4

194

1,2-Benzenedicarboxylic acid, dimethyl ester

3

7.667

0.44

C17 H36

240

Tetradecane, 2,6,10-trimethyl

4

7.750

0.49

C12 H24

168

Cyclododecane

5

7.909

0.86

C13 H28

184

Nonane, 3-methyl-5-propy

6

8.015

2.34

C18 H38

254

Heptadecane, 8-methyl

7

8.706

0.43

C18 H38

254

Heptadecane, 8-methyl

8

9.666

8.44

C12 H14 O4

222

1,2-Benzenedicarboxylic acid, diethyl ester

9

9.779

1.35

C11 H23 BR

234

1-Bromo-2-Methyl-Decan

10

9.931

1.56

C12 H14 O4

222

1,2-Benzenedicarboxylic acid, diethyl ester

11

11.007

0.30

C18 H38

254

Octadecane

12

12.153

0.47

C18 H30 O

262

2-tert-Butyl-4-(2,4,4-trimethylpent-2-yl)phenol

13

13.055

0.46

C16 H34

242

1-Hexadecanol

14

13.875

0.59

C16 H32 O2

256

Hexadecanoic acid

15

14.065

0.58

C16 H22 O4

278

1,2-Benzenedicarboxylic acid, dibutyl ester

16

15.084

0.50

C22 H38 O

318

2,4-Dioctylphenol

17

15.149

0.57

C15 H32 O

228

1-Pentadecanol

18

15.300

0.54

C33H48O4

508

Bis(2,4-Ditert-Butylphenyl) Penta

19

21.603

1.05

C24 H38 O4

390

1,2-Benzenedicarboxylic acid, dioctyl ester

20

22.742

75.07

C24 H38 O4

390

1,2-Benzenedicarboxylic acid, dioctyl ester

The highest peak area % was 1,2-Benzenedicarboxylic acid, dioctyl ester (75.07%) while the lowest peak % was Octadecane (0.30%).

Table 2: Activity of Components identified in sample using GC-MS

S.No

Molecular name

Biological activities**

1

Tetradecane

Antimicrobial, antifungal,

Nematicidal

2

Heptadecane, 8-methyl

Antifungal, Antibacterial,

Antioxidant

3

1,2-Benzenedicarboxylic acid, diethyl ester

Plasticizers

4

Hexadecanoic Acid

Antioxidant, Hypocholesterolemic

Nematicide, Pesticide, Lubricant, Antiandrogenic,

Flavor, Hemolytic

5-Alpha reductase inhibitor

5

Octadecane, dibutyl ester

Used as a plasticizer for vinyl foams, which are often used as floor tiles. Other uses are in traffic cones, food conveyor belts, and artificial leather

6

Octadecane

Lubricants, anticorrosion

agents

7

1,2-Benzenedicarboxylic Acid, Dioctyl Ester

Antimicrobial

Antifouling

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Figure 3: GC-MS Chromatogram of Boerhavia diffusa Ethanolic extract

GC-MS Analysis

By Shimadzu 2010 plus comprising AOC-20i autosampler GC MS analysis was carried out. Gas chromatograph employs the following conditions by interfacing mass spectrometer instrument: column RTX 5Ms with Column diameter is 0.32mm, column length is 30m, column thickness 0.50µm. It operates at 70eV electron impact mode; A carrier gas called Helium gas (99.999%) was used at a constant flow of 1.73 ml /min. An injection volume of 0.5 µI was employed with a ratio of 10:1.

The temperature for the injector is 270 ºC and for ion-source temperature 200 ºC. The oven temperature was programmed from 40 ºC (isothermal for 2 min), with an increase of 8 ºC/min, to 150 ºC, then 8ºC/min to 250ºC, ending with a 20min isothermal at 280ºC. Mass spectra were taken at 70eV with a scan-interval of 0.5 seconds and fragments from 40 to 450 Da. The running time of total GC is 51.25min. The relative component of each component was calculated when it is compared to the average peak area to the total area. By adopted Software mass spectra was handled. And the chromatogram obtained was in Turbo Mass Version 5.2.0.

Identification of components

By using the database of National Institute Standard and Technology (NIST05s), the GCMS interpretation was done because it has more than 62,000 patterns. A comparison was made between the unknown component spectrum and the known component spectrum. The structure, molecular weight and name of the components of the test materials were determined.

Results and Discussion

Identification of Bioactive compounds in Boerhavia diffusa Extract by GC-MS analysis

The GC-MS profile of plant Boerhavia diffusa showed the presence of 20 compounds. The active principles with their probability, molecular formula, molecular weight (MW) and area (%) are presented in Table 1 and Figure 3. The GC-MS output of Boerhavia diffusa showed two major peaks at retention times 9.66 and 22.74. The prevailing compounds are 1, 2-Benzenedicarboxylic acid, diethyl ester, Hexadecanoic Acid, 1, 2-Benzenedicarboxylic acid, dibutyl ester and 1, 2-Benzenedicarboxylic Acid, Dioctyl Ester were found in this plant Boerhavia diffusa. GC-MS is a cost-effective method but can able to cure many human diseases. The plant Boerhavia diffusa has many pharmacological activities since they act as steroidal lactones (Pooja, Lal, & Verma, 2017).

Biological activity

The biological activity of compound represented in Table 2. 1, 2-Benzenedicarboxylic acid, and diethyl ester was used in Plasticizers. 1, 2-Benzenedicarboxylic Acid reduces oxidative stress and able to cure neurodegenerative disorders (Choi et al., 2009).

Hexadecanoic acid is palmitic acid and has some biological activities such as anti-inflammatory effect, antioxidants, hypocholesterolemic, nematicide, pesticide, lubricant, hemolytic inhibitor and antiandrogenic.

Hexadecanoic acid is a saturated fatty acid shows antibacterial and antifungal activities (Aparna et al., 2012). Octadecane is the long-chain alkanes and acts as a lubricant and anticorrosion agents (Ghavipanjeh, Pazouki, Rad, & Hosseinnia, 2015).

Heptadecane acts as an antifungal and antimicrobial agents (Rao et al., 2016). 1-Pentadecanol has antimicrobial activity which can be analysed by selected microorganisms (Stein, Kimmel, Marincola, & Klemm, 2000).

1, 2- Benzenedicarboxylic acid, dibutyl ester used as a plasticizer for vinyl foams, which are often used as floor tiles. 1, 2-Benzenedicarboxylic Acid, Dioctyl Ester used for Antimicrobial, Antifouling.

The presence of various bioactive compounds justifies the use of the Boerhavia diffusa for various ailments by traditional practitioners. Solid cyclic hydrocarbon at room temperature is the Cyclodecane and this compound has ability to break stone (Gielen, Mélotte, Atassi, & Willem, 1989). Nonane shows increasing antitumor activity and has ability to cure cancer which is a dreadful disease (Kubo, Muroi, & Kubo, 1995).

Conclusion

20 different phytocompounds were found in the plant Boerhavia diffusa and their significance in pharmacological actions. Study also recommended the species as a plant of phytopharmaceutical importance due to the presence of biologically active compounds that my serve as candidates for new drugs in the treatment and prevention of many livestock diseases. A detailed study of the various compounds present in Boerhavia diffusa and their pharmaceutical importance requires to be carried out such that a drug with multiple effects can be made available in future studies.