Punica granatum root (s): Phytocompounds analysis, Anti-oxidant and Anti-microbial activity

 

Vignesh Balaji. E^*, Tamil Selvan. A, Srinivasan. A, Nandhini. S

 

ABSTRACT:

Punica granatum (PG) root (s) commonly known as pomegranate is a member of the monogeneric family, Punicaceae and is found throughout the world. Its phytocompounds possess numerous biological and toxicological properties including anti-oxidant, anti-inflammatory, anticancer and anti-angiogenesis activities. The present study was aimed to investigate anti-oxidant activity. Antioxidant potential was evaluated by the percentage inhibition of free radicals production by hydrogen peroxide method, which showed concentration dependent percentage inhibition of free radicals in both the methods. Also, the extract was analysed using HPTLC and GC-MS for the confirmation of active phyto compounds responsible for the desired biological activity. Furthermore, anti-microbial activity was done by disc diffusion and cup plate method. The zone of inhibition was evaluated by the test and standard sample.

 

 

 

INTRODUCTION:

Plant-based systems continue to play an essential role in healthcare, and their use by different cultures has been extensively documented¹. The World Health Organization (WHO) estimated in 1985 that approximately 65% of the population of the world predominately relied on plant-derived traditional medicines for their primary health care.

 

The pomegranate (Punica granatum) a fruit-bearing deciduous shrub in that grows between 5 and 10 m (16 and 33 ft) tall. Today, it is widely cultivated throughout the Middle East and Caucasus region, north and tropical Africa, South Asia, Central Asia, the drier parts of southeast Asia, and parts of the Mediterranean Basin. It is also cultivated in parts of Arizona and California. In the 20th and 21st centuries, it has become more common in the shops and markets of Europe and the Western Hemisphere⁹

 

Pomegranate seed oil contains punicic acid (65.3%), palmitic acid (4.8%), stearic acid (2.3%), oleic acid (6.3%), and linoleic acid (6.6%). Pomegranate ellagitannins are under preliminary research for their potential health benefits

 

MATERIALS AND METHODS:

Extraction of Plant Materials:

Weighed coarse powder used for the extraction by successive solvent extraction by Soxhlet apparatus¹⁰.

Hydroalcoholic (Ethanol 95% w/w) extract:

Marc obtained from the above extract was dried, extracted with hydroalcoholic mixture of ethanol and water in the ratioof 70:30 by using Soxhlet apparatus. The extraction was continued up to 24 hours. After completion of extraction, the solvent was filtered and removed by distillation under reduced pressure². Then it was stored in desiccator, freeze dried and used for the study.

 

PRELIMINARY PHYTOCHEMICAL EVALUATION:

The hydro alcoholic extracts of Punica granatum roots were subjected to phytochemical analysis for identification of plant constituents by chemical tests.

 

HPTLC ANALYSIS OF THE CRUDE EXTRACT:

Preparation of the Sample solution:

1gm of freeze dried hydroalcoholic extract powder was mixed with 10ml of methanol and completely dissolved and filtered using Whatman filter paper of grade 1. The filtrate was used for the HPTLC analysis.

 

Preparation of the Standard solution:

1gm of Quercetin powder was dissolved in 10ml of methanol and filtered using Whatman filter paper of grade 1. The filtrate was used for further studies.

 

Procedure:

TLC was performed on Silica gel 60F 254 TLC plates (E Merck, Germany) withToluene: Ethyl acetate: Methanol: Formic acid in the ratio of 5.5:3:1:0.5(v/v) as mobile phase. The bands were visualized in CAMAG UV Cabinet at 254nm and 366nm and scanned through WINCATS 4 software^3,4.

 

Table. No: 01. HPTLC values of Hydroalcoholic Extract of Punica granatum root (S)

S. No	Particulars	Values
1	LOD (ng/µl)	200
2	LOQ (ng/µl)	400 – 2000
3	r2 value	0.99882
4	r value	0.99941
5	Amount of Quercetin present in the extract (ng/gm)	<270.00

 

Fig.No. 01 GC – MS analysis of Hydroalcoholic extract of Punica granatum root(s)

ANTI-OXIDANT ACTIVITY:

Hydrogen peroxide scavenging activity:

A solution of hydrogen peroxide (40mM) was prepared in the phosphate buffer (pH–7.4). 1ml of sample in different concentration (100, 90, 80, 70, 60, 50, 40, 30, 20, 10µg/ml) in phosphate buffer were added to a hydrogen peroxide solution (0.6ml, 40mM) and 2.4ml of buffer solution⁵. Absorbance of hydrogen peroxide at 230nm was determined 10 minutes later against a blank solution containing the phosphate buffer without hydrogen peroxide.

 

                                                        Absorbance of sample

Percentage of antioxidant = -----------------------------------------   X 100

activity                                              Absorbance of control

 

ANTI-MICROBIAL ACTIVITY:

Preparation of test and standard solutions:

The stock solution of test compounds was prepared by dissolving the dried extracts at a concentration of 5 and 10 mg/ml in dimethylsulphoxide (DMSO) respectively. The stock solutions of reference standards (Ciprofloxacin) were prepared at a concentration of 0.6 mg/ml in sterile water. Antimicrobial activity was screened by adding the 0.05 ml of stock solution to each cup by micropipette.

 

RESULTS AND DISCUSSION:

Table.No:02. Extraction of Air-Dried Root(S) of Punica granatum (L)

Method of Extraction	Solvents	Colour	Consistency	Average value of extractive (% W/W)
Continuous Hot percolation by Soxhlet apparatus	Hydroethanolic (95%) Distilled water (70): Ethanol (30)	Dark brown	Liquid	22.74g (6.83)

 

PRELIMINARY PHYTOCHEMICAL ANALYSIS:

Table.No:03. Qualitative Analysis of The Crude Extracts of Punica granatum (L) Root (S)

Name of the Test	Petroleum ether extract	Hydro alcoholic extract
Carbohydrates	+	+
Reducing sugar	+	+
Proteins	-	-
Amino acids	-	+
Saponification	-	+
Cardiac glycosides	-	+
Anthraquinone glycosides	-	+
Saponins	+	+
Flavonoids	+	+
Alkaloids	-	+
Tannins and Phenolics	-	+
Steroids	-	+
Enzymes	-	-

(+)-Present, (-) – Absent

 

Fig.No:02 HPTLC 3D chromatogram of Hydroalcoholic extract of Punica granatum root(s)

 

 

Fig.No:03 HPTLC Chromatogram of Standard Quercetin

 

 

Fig.No:04 Finger Print Region Showing the Standard Compound  Quercetin with the Hydroalcoholic Extract

Table.No:04. GC-MSAnalysis of Hydroalcoholic Extract of Punica granatum (L) Root(S)

S. No	Retention Time	Peak area %	Name of the Compound	Molecular formula	Molecular weight	Chemical structure
1.	2.576	4.29	1-Propanethiol	C3H8S	76
2.	2.810	5.50	Ethane,1,1,1-triethoxy-O-acetic acid	C8H18O3	162
3.	4.887	6.76	1,1,3-Triethoxybutane	C10H22O3	190
4.	7.696	17.41	2-Isopropylpyrrolidine	C7H15N	113
5.	11.000	21.68	9-Aza-1- Methylbicyclo [3.3.1] nonan-3-one	C9H15NO	153
6.	23.595	10.29	n-Heptadecanol-1	C17H36O	256
7.	31.533	3.69	Xylitol, 1-O-octanoyl-	C13H26O6	278
8.	31.683	3.51	5-O-Methyl-d-gluconic acid dimethylamide	C9H19NO6	237
9.	32.546	6.17	Glyceroltricaprylate	C27H50O6	470
10.	35.089	11.39	1,2-Benzenedicarboxylic Acid	C24H38O4	390

 

Table. No.05. Invitro Antibacterial activity of Hydroalcoholic extract of PG root (s)

S. No	Concentration (µg/ml)		Name of the Organisms	Zone of Inhibition (mm)
	Test	Standard (Ciprofloxacin)		Test	Standard (Ciprofloxacin)
1	50	5	Klebsiella pneumonia	17	9
2	50	5	Escherchia coli	8	10
3	50	5	Bacillus subtilis	11	13
4	50	5	Staphylococcus aureus	19	27

 

 

Fig.No.05. Invitro Antioxidant Activity by Hydrogen Peroxide Scavenging Method

 

 

Fig.No: 06 Klebsiella pneumonia

 

 

Fig.No: 07 Escherchia coli

 

 

Fig.No: 08 Bacillus subtilis

 

 

Fig.No: 09 Staphylococcus aureus

CONCLUSION:

From this experimental study showed that the alcoholic extract of Punica granatum exhibited significant anti-oxidant activity against hydrogen peroxide. Furthermore, the presence of Phyto-compounds was revealed by HPTLC and GC-MS analysis^7,8. The ethanolic extract of Punica granatum root(s) contains the maximum amount of free radical scavenging action in the highest concentration (10mcg/ml). Also, in the HPTLC study, Quercetin as a bioflavonoid was identified and various Phyto ingredients was existing in the GC-MS analysis. In the anti-microbial study, the PG extract was compared with the standard (ciprofloxacin) by using both gram-positive and gram-negative organisms. The zone of inhibition was confirmed by cup plate and disc diffusion method.

 

CONFLICT OF INTEREST:

There is no any conflict of interest.

 

ACKNOWLEDGEMENT:

The author expresses their sincere thanks to the management of the PSG College of Pharmacy, Coimbatore, for encouraging and providing research facilities.

 

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Received on 24.06.2019                 Accepted on 31.07.2019

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Asian J. Pharm. Ana. 2019; 9(3):123-127.