Phytochemical Screening and Antimicrobial Activity of Ziziphus oenoplia Seed Extract

 

Anbu S¹, Boomiga S², Suresh A¹, Padma J³*

¹Department of Biochemistry, Shanmuga Industries Arts and Science College, Thiruvannamalai – 606601.

²PG and Research Department of Biochemistry, Scared Heart College (Autonomous),

Tirupattur, Vellore - 635 601.

³Government Seed Testing Laboratory, Venkiggal, Thiruvannamalai – 606604.

 

ABSTRACT:

The present aimed to analysis the phytochemical and pharmacological activities of Ziziphus oenoplia seed extract. Hence the ethanolic extract was prepared and analyzed Gas chromatography-mass spectrometry (GC-MS) and Fourier Transform Infrared Spectrophotometer (FTIR). The GC-MS analysis of Ziziphus oenoplia showed 24 major peaks revealing the presence of 16 major phytochemical compounds in the Ziziphus oenoplia extract. Further, the FTIR analysis also confirmed the presence of alcohols, aromatic compound, alkanes, aldehydes, ketones, alkenes, amines, amides, nitro compounds, carboxylic acids, ethers and ester. Antimicrobial activity was tested against S. typhi, S. aureus and E.coli. The extracts showed significant inhibition towards the selective microbes. The identified compounds are further in needed to study in their pharmacological activities. Hence, the further studies are warranted. It could be expected Ziziphus oenoplia Seeds extract might be great helpful for human ailments.

 

 

 

INTRODUCTION:

Plants play a vital function in the development of new drugs to be used for treatment of various illnesses. According to World Health Organization traditional medicine from plant extracts provide health coverage over 80% of total world population. 80% of rural population totally depends on medicinal plants and its derived products. Nowadays, peoples are moving towards the nature and its derived products as remedies in the disorders of various disease to control, suppress and prevention. Indian are very ancient followers, the system has been well advanced. There are thousands of Chemical compounds are formulated from plants for infectious diseases with potential activities. Although, a large numbers of phytochemicals are identified in a particular plant it, but it is undefined to use entire plant as medicine.¹

 

 

 

Fourier transform infrared (FTIR) spectroscopy is an analytical technique, provides a vibrant picture of the phytochemical composition in an organic and non-organic samples at a given particular time. It is possible to detect the minor changes in the primary and secondary metabolites in samples by observing the IR spectra.² FTIR is engaged to explain the structure of unknown compounds and the strength of absorption spectra associated with molecular composition or chemical functional groups.³ FTIR is being useful in structures of plant metabolites and its characterization of plant and microbial species.⁴

 

Gas liquid chromatography jointly with specific detection schemes is relatively expensive and often laborious techniques. Analysis of chemicals has become easier and more cost-effective. Gas chromatography-mass spectrometry (GC-MS) analysis can recognize compounds even less than 1mg in plant extract.⁵ GC-MS is simple, rapid tests and less cost-effective for detecting phytocomponents. Nowadays GC-MS technique has been progressively increase working to identify the secondary metabolites present in the herbs. This technique has been proven valuable method for the analysis and detection of essential oil, alcohols, acids, esters, alkaloids, steroids, amino and nitro compounds.⁶

 

In an atoms there are number of unpaired number of electrons produced as result of various metabolic reactions. The unpaired electrons are so are called free radicals and are of highly reactive. These unpaired electrons can start up chains of reactions in biological system. The above free radicals can give raise to the production of reactive oxygen spices. They are highly toxic to the cells and permissible to destroy the macromolecules including carbohydrates, proteins, lipids, Nucleic acids and so on.⁷ The toxic radicals are tightly controlled by endo/exogenous antioxidants and maintenance in tightly controlled manner under normal physiological conditions. The various endogenous antioxidants enzymes and proteins play a major role in the regulation of productions free radicals and its destruction. The instability between these two conditions leads to oxidative stress.

 

Diabetes, cardiovascular diseases, Cancers, neurological disorders, stroke, asthma, liver injury etc., have developed as result of imbalance maintenance. Further it can arise as a result of age-related degenerative diseases and other non-communicable disease. Exogenous antioxidants foods source, can deferral or hinder the initiation or propagation of oxidative reactions.⁸ Numerous synthetic antioxidants are proven effective with lesser side effect. Therefore, today the researchers are interested in medicinal plants for its evaluation of antioxidant containing phytochemicals, which comprises phenols, flavonoids and tannins and their potential role in the prevention of human illnesses.⁹

 

Ziziphus oenoplia is known as small-fruited jujube. Its tamil name is suraimullu. The height of Ziziphus oenoplia is 1.5m. It is spreading and climbling, throny shrub. The fruit of ziziphus oenoplia is sparkly when it becomes ripe that contains only a single seed. There is a long history of using this herbal plant as medicine.¹⁰ It produces ziziphines, cyclopeptide alkaloids. The leaves are being useful in dressing for wounds and the stem bark is used for sore throat. The saliva production can be increased by taking the leaves and fruits. Human wellbeing can be brought by knowing the knowledge of the plant and too develop new medicines. The flower is scandent shrubs, and throns. Ziziphus oenoplia leaves are an alternate, ovate-lancedate, and oblique. Ziziphus oenoplia flower is green in sessile axillary cymes. Fruit was single seed and seed is oval in shape.¹¹ Based on the literature review, there were no reports on the phytochemical screening analysis of seed extracts of Z. oenopliais, hence, it is interested to know the presence of phytochemicals and its pharmacological activities.

 

MATERIALS AND METHODS:

Preparation of Plant Extracts:

The seeds of Ziziphus oenoplia were collected from Nattrampalli Village, Tirupattur, Tamil Nadu, and India. The plant specimen was identified and authenticated in the department of Botany, Annamalai University, Annamalai Nagar, Tamilnadu. The seeds were allowed to shade dried and powdered. The crude plant extract was prepared by using Soxhlet extraction method. 15g of powdered plant material was extracted with 200ml of 60% ethanol. The extracts was filtered separately, and evaporated to dryness. Further the dried extract was maintained in an incubator and used to analyze the phytochemical screening, GC- MS analysis, FTIR and antibacterial activity.

 

Chemicals:

Ethanol, sodium hydroxide, acetic acid and benzene were obtained from Hi-Media Chemical Pvt. Ltd, Mumbai. All the chemicals and reagents used in the present investigation were of analytical reagent grade and glass distilled water used throughout the study.

 

Phytochemical screening:

Phytochemical screening has been carried out by the following methods.¹²

 

Antibacterial activity:

Antibacterial tests will be carried out by the agar well diffusion method using the suspension spread on nutrient agar. Dip the swab into the broth culture of the organism. Gently squeeze the swab against inside of the tube to remove excess fluid. Use the swab to streak agar plate a nutrient agar plate for a lawn of growth. sing the well culture cut a well to load the samples and was placed on the well, in the range of 50µl and 100µl. Then antibiotic was used as the control (Streptomycin and Chlorophenical (50µg/mL). Then the incubated plates were incubated at 37°c for 24 hrs, the zone of inhibition was observed.¹³

 

GC- MS Analysis:

GC-MS analysis was carried out on a GC clarus 500 Perkin Elmer system comprising a AOC-20i autosampler and gas chromatograph interfaced to a mass spectrometer instrument employing the following conditions: column Elite-1 fused silica capillary column (30 x 0.25mm ID x 1μMdf, composed of 100% Dimethyl polydiloxane), operating in electron impact mode at 70eV; Helium gas (99.999%) was used as carrier gas at a constant flow of 1ml/min and an injection volume of 0.5μI was employed (split ratio of 10:1) injector temperature 250ºC; ion-source temperature 280ºC. The oven temperature was programmed from 110ºC (isothermal for 2 min), with an increase of 10ºC/min, to 200ºC, then 5ºC/min to 280ºC, ending with a 9min isothermal at 280ºC. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds and fragments from 40 to 450 Da. Total GC running time is 36min. min. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was a TurboMass Ver 5.2.0.¹⁴

 

FTIR Analysis:

FTIR analysis of phytochemicals was carried out by the method described elsewhere.¹⁵

 

RESULTS AND DISCUSSION:

Qualitative analysis of phytochemicals:

The preliminary phytochemical studies based on biochemical protocols and its results were given in Figure 1a&b. The results have confirmed that the plant extract of Ziziphus oenoplia showed positive results for various phytochemical components and the same were recorded in Table 1.

 

 

Figure - 1a: Qualitative analysis of phytochemicals

 

Figure-1b: Qualitative analysis of phytochemicals

 

It can be concluded that the plant contains numerous phytochemicals predominantly in the present seed extract of Ziziphus oenoplia. The presence of above phytochemicals might be responsible for the various medicinal uses of Ziziphus oenoplia plant and its fruits. The current results are corroborated with various previous reports. The plant derived secondary metabolites are plays a major role in the treatment of various ailments in siddha. Previous report on phytochemical screening of methanolic extract of root and stem of plant extract have showed the presence of phenols, flavonoids, saponin, tannins and alkaloids, the same plant extract were also subjected its antioxidant activity (Leelavathi and Udayasri, 2018). The secondary metabolites acts as antioxidant and metabolic regulator and thus play a protective role. Previous reports have shown that the plant contains secondary metabolities have shown antioxidants, antimicrobial, antiviral, antimalarial, antiasthma, and anticancer and so on. Due to the presence of biological activities these phytochemicals are being potential uses as drugs.¹⁶

 

Table 1: Shows the qualitative analysis of phytochemicals

S. No	Phytochemical	Test	Observation	Results
1.	Alkaloids	Wagner’s	Reddish brown precipitate	+
2	Flavonoids	NaOH (Alkaline reagent)	Intense yellow colour	+
3	Glycosides	Conc.H2SO4	Brown ring	+
		Molish’s	Violet ring	+
4.	Phenols	Ellagic Acid	Muddy precipitate	+
		Ferric Chloride	Bluish black colour	+
		Lead Acetate	Yellow colour precipitate	+
5.	Protein and Amino acid	Ninhydrin	-	-
		Xanthoprotein	-	-
6.	Saponins	Foam	Layer of foam	+
7.	Steroids	Salkowski	Greenish yellow	+
8.	Tannins	Gelatin	precipitate	+
		Ferric Chloride	Blue colour	+
9.	Quinones	Sulphuric acid	Red colour	+
10.	Terpenoids	Copper acetate	Emerald green	+

 

 

Antibacterial activity of Ziziphus oenoplia seed extracts

 

Figure-2: Antibacterial activity of Ziziphus oenoplia seed extract against selective microbes

 

The antibacterial activity of Ziziphus oenoplia seed extract was given in the figure 2. There were 3 species chosen for the study, which includes S. typhi, S. aureus and E.coli. The extracts showed significant inhibition towards the selective microbes. Among the inhibition the plant extract showed more potent inhibition against the E.coli and having the moderate inhibition against the two other species (S. typhi and S. aureus). Plants are being useful for the infectious disease, since long back.^17,18 The problem of microbial resistance becomes serious concern. Search of new compound with less side effects are great needful. Previous report has shown that there are many plant extracts having significant antimicrobial properties against a selective species. Our results are also in concurrent with the previous reported.^19,20

 

GCMS Analysis:

The GC-MS analysis of Ziziphus oenoplia showed 24 major peaks revealing the presence of 16 major phytochemical compounds in the ZoS extract. The 16 phytochemical compounds present in the Ziziphus oenoplia extract were identified by matching the peaks obtained with the NIST spectral library. The compounds identified are listed in the Table 2 - with compound name, with its retention time, molecular formula, molecular weight and peak area %.

 

The 16 major phytoconstituent’s compound retained from Ziziphus oenoplia was namely Ethyl 3-hydroxytetracosanoate1,2-Benzenedicarboxylic Acid, Bis (2-Methylpropyl) Ester, n-Hexadecanoic acid, n-Hexadecanoicacid, Hexadecanoic Acid, Ethyl Ester, 9,12-Octadecadienoic acid (Z,Z)-, Octadec-9-Enoic Acid, Octadecanoic acid, Hexane, 3,3-Dimethyl, Spiro[Cyclopentane-1,2'(1'h)-Quinoxaline], 3'-(4-Morpholinyl)-6',8'-Dinitro- Hexane, 2,3,4-Trimethyl, 1,6,10,14,18,22-Tetracosahexaen-3-ol, 2,6,10,15,19,23-hexamethyl-, (all-E), Dodecyl nonyl ether (Methyl Behenate), 5-Hydroxymethylfurfural, 1,3-Dioxolane, 2-Ethyl-2-Isopropyl-4,5-Dimethyl, n-Propyl cyanoacetate and (Z)-4-Nonenyl Acetate were respectively.

 

Previous report has shown that the leaves of K. pinnata revealed that the incidence of Hexadenoic acid compound and in M. officinalis 17 compounds with n-Hexadecanoic acid and Octadecanoic acid was identified as the major compounds in the leaves of C. collinus. GC-MS analysis of ethyl acetate extract of G. umbrosus reported that the presence of n-Hexadecanoic acid. Hexadecanoic acid, 9, 12 - Octadecadienoic acid, n-hexadecanoic acid, 9, 12, 15-Octadecatrienoic acid, Squalene and Phytol were identified in the ethanol leaf extract of Aloe verA. ¹⁴

 

The source of many plants can be often identified from the peak pattern of the chromatograms obtained directly from headspace analysis. Likewise particular qualitative and quantitative outlines from a GC analysis will show all the compounds in the leaf extract. Previous report with plants extracts have many biological properties which can be used in various purposed to treat many diseases. The compounds identified by qualitative analysis and GCMS screening have many uses in medical field. Each compounds identified have their unique character to treat various diseases. Further studies needed to reveal its importance in specific field to treat the diseases properties.²¹

 

Table 2: Shows the presence of phytochemicals in Ziziphus oenoplia seed Extracts

Bioactive compounds	Molecular formula	Molecular Weight	Retention time	Area (%)
5-Hydroxymethylfurfural(Sugars)	C6H6O3	126.11	5.764	1.43
1,3-dioxolane, 2-ethyl-2-isopropyl-4,5-dimethyl-	C7H14O2	130.18	6.427	1.06
n-Propyl cyanoacetate	C6H9NO2	127.14	8.49	1.94
(Z)-4-nonenyl acetate	C11H20O2	184.27	8.872	8.63
3-hexen-1-ol	C6H12O	100.16	10.291	5.65
Pentane, 2,3,3-trimethyl-	‎C8H18	‎114.23	12.366	5.41
beta.-D-Glucopyranoside, methyl	‎C7H14O6	194.18	16.093	6.51
1,3,4,5-Tetrahydroxy-Cyclohexanecarboxylic Acid	C7H12O6	192.17	19.895	16.48
Ethyl 3-hydroxytetracosanoate	C26H52O3	412.68	21.058	2.49
1,2-Benzenedicarboxylic Acid, Bis (2-Methylpropyl) Ester	C16H22O4	278.34	21.134	11.13
n-Hexadecanoicacid	C16H32O2	‎256.42	21.322	4.34
Hexadecanoic Acid, Ethyl Ester	C18H36O2	284.47	21.375	0.23
9,12-Octadecadienoic acid (Z,Z	C18H32O2	280.41	21.41	0.27
Octadec-9-Enoic Acid	‎C18H34O2	282.5	21.735	0.36
Octadecanoic acid	C18H36O2	298.5	24.937	0.35
Hexane, 3,3-Dimethyl-	C8H18	114.23	26.682	0.29
Spiro[Cyclopentane-1,2'(1'h)-Quinoxaline], 3'-(4-Morpholinyl)-6',8'-Dinitro-	C6H19N5O5	361.11	27.152	0.24
Hexane, 2,3,4-Trimethyl	C9H20	128.25	30.345	1.43
1,6,10,14,18,22-Tetracosahexaen-3-ol, 2,6,10,15,19,23-hexamethyl-, (all-E)-	C30H50O	426.71	33.166	1.94
Dodecyl nonyl ether (Methyl Behenate)	C21H44O	312.6	34.495	8.63
5-Hydroxymethylfurfural	C6H6O3	126.11	35.773	5.65
1,3-Dioxolane, 2-Ethyl-2-Isopropyl-4,5-Dimethyl	C7H14O2	130.18	37.004	5.41
n-Propyl cyanoacetate	C6H9NO2	127.14	38.196	6.51
(Z)-4-Nonenyl Acetate	C11H20O2	184.27	38.342	16.48

 

FTIR spectroscopy analysis:

 

Figure - 5: FTIR analysis of Ziziphus oenoplia seed extracts

 

Table 3: FTIR peak values of fruit extract

S. No	Peak value	Functional groups
1	3879.74	O-H stretching
2	3371.85	O-H stretching
3	2992.15	C-H stretching
4	2927.42	C-H stretching
5	2743.79	C=O stretching
6	2534.85	C=O stretching
7	2131.30	C=C stretching
8	1640.23	C = N stretching imine/oxime or C = O stretching conjugated ketone or alkenes
9	1426.85	Aromatic
10	1307.12	C-O stretching vibration, presence of alcohols, carboxylic acids, esters, ethers
11	1118.01	C-N stretching amine
12	818.34	Amine

 

Fourier transform infrared spectroscopy is an important analytical tool which the metabolic composition of plants and non-plants source at a given time (Saravana Kumar et al., 2015). Based on the peak value in an infrared region of the Ziziphus oenoplia (ethanolic) extracts were shown in figure 4. Totally 11 sharp transmittance peak were observed at 3879.74 & 3371.85 (O-H stretching), 2992.15 (C-H stretching), 2927.42 (C-H stretching), 2743.79 (C=O stretching), 2131.30 (C=C stretching), 1640.23 (C=O stretching), 1426.85 (C=O stretching), 1307.12 (C-O stretching) and 1118.01 & 818.34 (C-H Bending). These peaks were revealed the presence of alcohols, aromatic compound, alkanes, aldehydes, ketones, alkenes, amines, amides, nitro compounds, carboxylic acids, ethers and ester.

 

SUMMARY AND CONCLUSION:

The GC-MS analysis of Ziziphus oenoplia showed 24 major peaks revealing the presence of 16 major phytochemical compounds. Further, the FTIR confirmed the presence of alcohols, aromatic compound, alkanes, aldehydes, ketones, alkenes, amines, amides, nitro compounds, carboxylic acids, ethers and ester. Antimicrobial activity against S. typhi, S. aureus and E.coli were tested. The extracts showed significant inhibition towards the selective microbes. The identified compounds are further in needed of study for their pharmacological activities. Hence, the further studies are warranted. It could be expected Ziziphus oenoplia Seeds extract can be beneficial for human ailments.

 

ACKNOWLEDGEMENTS:

Authors are grateful to Principal and The Management of Sacred Heart College (Autonomous), Tirupattur for their support during the study

 

REFERENCE:

1.      Shukla A, Garg S, Garg A, Mourya P, Jain CP. Investigations on hydroalcoholic extract of Zizyphus oenoplis for analgesic and anti- nociceptive activity. Asian Pacific J Trop Med. 2016; 1: 283-288

2.      Shanmuga Priya. S, Aruna Sharmili. S, Anbumalarmathi. J, Umamaheswari. K. Evaluation of Phytochemical Constituents, In vitro Antimicrobial, Antioxidant, FT-IR and GC-MS Studies of Leaves of Solanum torvum, Rhizome of Acorus calamus and Whole Plant of Mollugo pentaphylla. Research J. Pharm. and Tech. 2017; 10(2): 592-600.

3.      Archana Kulkarni, Nasreen Jan, Seema Nimbarte. GC-MS, FT-IR and NMR Spectroscopy Analysis for Metabolome Profiling of Thyme Oil. Asian J. Research Chem. 2013: 6(10); 945-949.

4.      Yang J, Yen HCE. Early salt stress effects on the changes in chemical composition in leaves of ice plant and Arabidopsis - A Fourier Transform Infrared Spectroscopy study. J Plant Physiol. 2002; 130:1032-1042

5.      Edwin Jose B, PanneerSelvam P. Identification of Phytochemical Constituents in the Leaf Extracts of Azima tetracantha Lam using Gas Chromatography-Mass Spectrometry (GC-MS) analysis and Antioxidant Activity. Asian J. Research Chem. 2018; 11(6): 857-862.

6.      Muthulakshmi A, Margret J and Mohan VR. GC-MS analysis of bioactive components of Feroniaele phantumcorrea (Rutaceae). App. pharmac. Sci. 2012; (2):69-74.

7.      Petersen RC. Free-radicals and advanced chemistries involved in cell membrane organization influence oxygen diffusion and pathology treatment. AIMS Biophys. 4(2): 240–283 Pharmacology. 2017;. 2(1): 15-18

8.      Swetha Sunkar, Akshaya. A, Aarthi. B, Valli Nachiyar. C, Prakash. P. Phytochemical analysis and isolation of endophytic bacteria from Bauhinia purpurea. Research J. Pharm. and Tech 2018; 11(5):1867-1876.

9.      Senguttuvan J, Paulsamy S, Karthika K. Phytochemical analysis and evaluation of leaf and root parts of the medicinal herb, Hypochaeris radicata L. for in vitro antioxidant activities. Asian Pac J Trop Biomed. 2014; 4 (1): S359-S367

10.   Ramalingam R, Madhavi BB, Nath AR, Duganath N, Sri UE, Banji D. Invitro antidenaturation and antibacterial activities of Ziziphus oenoplia. Scholars Research Library, Der Pharmacia. Lettre. 2010; 2(1): 87-93

11.   Rao V, Rawat AKS, Singh Anil P. Hepato protective potential of Ethanolic extract of (L) Mill roots against Ziziphus oenoplia antitubercular drugs induced hepato toxicity in experimental models. Asia Pacific J Trop Med 2012; 4: 283-288

12.   Visweswari G. Christopher R and Rajendra W. Phytochemical screening of active secondary metabolites present in withania somnifera root: role in traditional medicine. IJPSR. 2013; 4:7: 2770-2776.

13.   Gokila Devi, T. Nagaraja Suryadevara, Divyabharathi, Gopinath, L. R. Venkatasathya Sai Appala Raju Velaga, Ponmurugan P. Efficacy of Moringa oleifera’s Therapeutic Compounds and its Antimicrobial Activity. Research J. Pharm. and Tech. 2020; 13(8):3867-3872.

14.   Kilimozhi D, Parthasarathy V, Manavalan R. Active Principles Determination by GC/MS in Delonix Elata and Clerodendrum Phlomidis. Asian J. Research Chem. 2009; 2(3): 344-348.

15.   Saraswathi. K, Sivaraj. C, Jenifer. A, Dhivya. M, Arumugam. P. Antioxidant, Antibacterial activities, GCMS and FTIR Analysis of Ethanol bark extract of Capparis sepiaria L. Research J. Pharm. and Tech 2020; 13(5): 2144-2150.

16.   Varadarajan, P., Rathinaswamy, G. and Asirvatahm, D. Antimicrobial properties and phytochemical constituents of rheo discolor. Ethnobotanical Leaflet. 2008; 12: 841-845

17.   Mitesh D Phale, Dipti Korgaonkar. Current Advance Analytical Techniques: A Review. Asian J. Research Chem. 2009:2(3); 235-238.

18.   Mariadoss AVA, Ramachandran V, Shalini V, Agilan B, Franklin JH, Sanjay K, Alaa YG, Tawfiq MA, Ernest D. Green synthesis, characterization and antibacterial activity of silver nanoparticles by Malus domestica and its cytotoxic effect on (MCF-7) cell line. Microb Pathog. 2019 ;135:103609.

19.   Duraipandiyan V, Ayyanar M, Ignacimuthu S. Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India,” BMC Comp Alt Med. 2006; 6:35

20.   Gao, Y., Arokia Vijaya Anand, M., Ramachandran, V. et al. Biofabrication of Zinc Oxide Nanoparticles from Aspergillus niger, Their Antioxidant, Antimicrobial and Anticancer Activity. J Clust Sci 2019; 30: 937–946.

21.   Rukshana MS, Doss A and Kumari Pushpa Rani TP. Phytochemical Screening and GC-MS Analysis of Leaf Extract of Pergularia daemia (Forssk) Chiov. Asian J Plant Sci Res. 2017; 7(1): 9-15.

 

 

 

 

 

 

Accepted on 25.07.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(2):615-620.