1. INTRODUCTION:

According to World Health Organization, “a medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes, or which are precursors for chemo pharmaceutical Semi-synthesis”. The traditional systems of medicine are still considered as a great knowledge based in herbal medicines¹.

The medicinal plants have regained a wide recognition to combat infectious diseases due to the failure of modern drugs against various chronic diseases along with increasing costs and side effects associated with them.

Emergence of multi-drug resistant strains of pathogenic bacteria and higher safety margins also contribute towards increasing interest in traditional medicine. Herbs have recently attracted attention as health beneficial foods and as source materials for drug development. Herbal medicines derived from plant extracts are being increasingly utilized to treat a wide variety of clinical diseases.^2,3.

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), which is the top cause of death from infectious. It has been a huge threat to public health worldwide for a long time, with about 10 million people infected each. The emergence of multidrug resistant TB (MDR-TB), extensively drug-resistant TB (XDR-TB), and HIV co-infection made it more challenging to prevent the prevalence of TB infections. Tuberculosis remains a global health problem with an enormous burden of disease, estimated at 10.4 million new cases in 2015, of which 10% were among children and 12% involved human immunodeficiency virus (HIV) coinfection. In 2015, there were an estimated 1.8 million deaths due to tuberculosis, including HIV-associated tuberculosis deaths, making tuberculosis the leading cause of death from an infectious disease.⁴. Latent Mycobacterium tuberculosis infection is the reservoir of the tuberculosis epidemic. The global burden of M. tuberculosis infection has recently been reestimated at 24% ⁵.

Traditional anti-TB drugs such as isoniazid, rifampicin, ethambutol, and pyrazinamide have been the front line in the battle against TB for many years. Although dramatic progresses have been achieved, there is still an urgent need for new anti-TB agents that are highly active with low toxicity due to the increasing drug resistance problems. Therefore, novel drug targets for developing new anti-TB drugs are still necessary⁶. The emergence of multidrug resistant TB (MDR-TB), extensively drug-resistant TB (XDR-TB), and HIV co-infection made it more challenging to prevent the prevalence of TB infections^7,8. Antibiotic resistance is an emerging problem in these days. Multiple drug resistance has developed due to the indiscriminate use of antimicrobial drugs which are commonly used in the treatment of infectious diseases. Antibiotics causes many adverse effects on the host including hypersensitivity, immune-suppression and allergic reactions. The necessities for the importance of new and effective antimicrobial substances from different plants is to control the infections and to overcome the bacterial resistance without any side effects^{9, 10.}

Ziziphus nummularia (W and A) a common synonym of Ziziphus rotundifolia (Jhar beri), belongs to family Rhamnaceae has numerous uses for animals, agriculture and medicine. It is used in traditional medicine of many Asian countries. Leaves are used as antipyretic, antiobesity, anti-inflammatory and against expectorant, scabies and boils^.11-13. Fruits are used as tonic, digestive, laxative, astringent, aphrodisiac, purifies blood and removes biliousness, thirst, vomiting, burning sensations, and used against loss of appetite, chronic fatigue, diarrhea, pharyngitis, bronchitis, burns, anemia, irritability hysteria ^14-16. The branches are often used for fencing the fields, the leaves are threshed out and used as fodder. The plant has been reported that it contains polysaccharides, peptide alkaloids, flavanoids, saponins, triterpenoids, and fatty acids. Phytoconstituents isolated from Zizyphus nummularia plant are Nummularogenin and zizynummin ^17,18

The literature survey reveals that most of the research work has been conducted on leaves, fruit and stem. The root of the plant remains unexplored for its phyto-chemical, bio-medical potential and their applications. So, it was decided to extract the powdered root and explore its bacterial resistance activity.

2. MATERIALS AND METHODS:

2.1 Plant collection:

The plant Ziziphus nummularia was collected fromVirudhunagar District, Tamil Nadu, India during the month of October and was authenticated by Botanist Dr. T. Lingakumar Professor, Department of Botany, ANJA college of arts and science, Sivakasi, Virudhunagar district.

2.2. Preparation of extract:

The roots were collected, washed with water to get free from debris and dried under shade. The dried roots were powdered by grinding and passed through sieve no 60. The powder was stored in a well closed air tight jar and used for further screening. About 300 gm of the dried powdered root of Ziziphus nummularia was defatted with 1000ml petroleum ether (60-80⁰C) for 24 hours by maceration. The solvent was removed by filtration and the marc was dried. To the dried marc 500ml of ethanol was added in a separate round bottom flask and the extraction was performed by using soxhlet apparatus (6hr). It was then filtered and filtrate was evaporated to a cohesive mass using rota vapour^19-21. The residue obtained was stored in the refrigerator and subjected to qualitative chemical analysis. The same process was carried out to get aqueous extract.

2.3. Phytochemical screening:

The Preliminary Phytochemical tests were performed on the various extracts of Ziziphus nummulariaroot for the identification of sterols, terpenoids, flavonoids, anthraquinone, sugar, glycosides, alkaloids, phenols, saponins and tannins as per standard procedure^22-25.

2.4. Antimicrobial activity:

The microbial cultures Gram positive organism such as Staphylococcus aureus, Streptococus thermophiles, gram negative organisms such as Escherichia.coli, Shigella dysenteriae were used for this study. All the bacterial cultures were obtained from Microbial Type Culture Collection & Gene Bank (MTCC), Sector 39-A, Chandigarh.

The agar well diffusion method was employed for the determination of antimicrobial activity of the extracts. About 20 ml of Muller Hinton agar medium for bacteria and potato dextrose agar for fungus was poured into sterilized Petri dishes and allowed to solidify. The agar medium was spread with 24hrs cultured 108 CFU/ml of microbial strains by a sterilized rod. The wells (6mm in diameter) were made in the agar plates using a sterilized cork borer. About 10,20,40,80 μgm/ml of the extracts were poured into the well using a sterile micropipette. Two common antibiotics Ciprofloxacin and Tetracycline (30µgm/ml) were used as reference drug, and corresponding solvents (ethanol, water) were used as positive control for the assay. Then the plates were incubated at 37±2°C for 24 hours for bacterial activity and 48 hours for fungal activity. The plates were observed for the zone formation around the wells. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well (in mm) including the well diameter ^26-29

2.5. Anti-Tuberculosis activity:

The anti-tuberculosis activity were assessed against Mycobacterium tuberculosis using Microplate Alamar Blue assay (MABA) as per standard procedure^30-33. This methodology is non-toxic, uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method.

Briefly, 200µl of sterile deionized water was added to all outer perimeter wells of sterile 96 wells plate to minimized evaporation of medium in the test wells during incubation. The 96 wells plate received 100µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate. The final drug concentrations tested were 100 to 0.2µg/ml. Plates were covered and sealed with parafilm and incubated at 37ºC for five days. The standard synthetic Anti-TB drug such as Pyrazinamide, Ciprofloxacin and Streptomycin were used. The bacterial strain used are M. tuberculosis (H37 RV strain): ATCC No- 27294.

After this time, 25µl of freshly prepared 1:1 mixture of Almar Blue reagent and 10% tween 80 was added to the plate and incubated for 24hrs. A blue color in the well was interpreted as no bacterial growth, and pink color was scored as growth. The Minimum inhibitory concentration (MIC) was noted.

3. RESULTS AND DISCUSSION:

3.1. Nature of extracts:

The quantity of the extracts obtained by using solvents (Aqueous and Ethanol) and its nature noted (Table-1).

Table 1 – Nature of The Extracts

S. No	Extracts	Colour	Consistency	Quantity (Gms)
1	Ethanol	ReddishBrown	Fine solid	20.97
2	Aqueous	Dark Brown	Semi solid	18.52

3.2. Phytochemical screening:

From the qualitative phytochemical screening study it has been observed that the extracts of Ziziphus nummularia root contains all the necessary primary and secondary metabolites such as sterols, tannins, saponins, carbohydrates, triterpenoids, proteins and flavonoid, except alkaloids and glycosides. The results were tabulated in the Table-2

Table 2-Preliminary Phytochemical Screening for The Extracts of Ziziphus nummularia ROOT

S. No	Test	Ethanolic extract	Aqueous extract
1.	Test for Carbohydrates
	a. Molisch’s test	+	+
	b. Fehling’s test	+	+
	c. Benedict’s test	+	+
2.	Test for Proteins
	a. Million’s test	-	-
	b. Biuret test	-	-
3.	Test for Alkaloids
	a. Mayer’s reagent	-	-
	b. Dragendorff’s reagent	+	-
	c. Hager’s reagent	+	-
	d. Wagner’s reagent	+	-
	e. Test for Purine group (Murexide test)	-	-
4.	Test Forglycosides
	a. Anthraquinone glycosides
	i) Borntrager’s test	-	-
	ii)Modified Borntrager’s test	-	-
	b. Cardiac glycosides
	i) Keller Killiani test	-	-
	c.Cyanogenetic glycosides	-	-
5.	Test For Saponins	+	+
6.	Test for Tannins
	Fecl3 test	+	+
7.	Test For Flavonoids
	a. Shinoda test	+	-
	b. Alkali test	+	+
	c. Acid test	+	-
	d.Ammonia test	+	+
8.	Test For Sterols
	a. Salkowski’s test	+	-
	b. Libermann-burchard’s test	+	-
9.	Test for Terpenoids	+	-
10.	Test for Mucilage	+	-

3.3. Antimicrobial activity

The antimicrobial activity for extracts of Ziziphus nummularia root and standard drug were screened by Agar well diffusion method. The zone of inhibition for different extracts at various concentrations against micro-organisms are noted. The ethanolic extract showed maximum zone of inhibition 24 mm at 80 mg/ml concentration against Staphylococci and minimum zone of inhibition 13 mm at 10 mg/ml concentration against E.coli Also certain microorganisms such as Shigella and Streptococci are resistance to ethanolic extracts of Ziziphus nummularia root Similarly the aqueous extract of Ziziphus nummularia root showed maximum zone of inhibition 14 mm at 80 mg/ml concentration against Staphylococci and minimum zone of inhibition 5 mm at 10 mg/ml concentration against E.coli The standard drug ciprofloxacin , tetracycline shown a zone of inhibition as 8mm and 11mm at 30 mg/ml (Table-3 and Fig 1).

Table 3: Ethanolic Extract of Ziziphus nummularia Zone of Inhibition (mm)

CONC mg/ml	*E. coli*		Shigella		*Streptococci*		*Staphylocooci*
CONC mg/ml	A. E	E. E	A. E	E. E	A.E	E.E	A.E	E.E
10	1	13	7	R	8	R	11	16
20	8	15	8	R	11	R	11	21
40	11	15	8	R	12	R	13	22
80	12	17	11	R	13	R	14	24
Cipro	(30mg/ml		8
Tetra	(30mg/ml		11

Aqueous extract – A.E. Ethanolic extract – E.E

Table 4-MIC for extracts of Ziziphus nummularia Root

S. No

Sample

0.8

μg/ml

1.6

μg/ml

3.12

μg/ml

6.25

μg/ml

12.5

μg/ml

100

μg/ml

Aqueous Extract

Ethanolic extract

Note: + Sensitive – Resistant

Table 5-Mic for Standard Drugs

S. No	Sample	0.2 μg/ml	0.4 μg/ml	0.8 μg/ml	1.6 μg/ml	3.12 μg/ml	6.25 μg/ml	12.5 μg/ml	25 μg/ml	50 μg/ml	100 μg/ml
1	Pyrazinamide	-	-	-	-	+	+	+	+	+	+
2	Ciprofloxacin	-	-	-	-	+	+	+	+	+	+
3	Streptomycin	-	-	-	-	-	+	+	+	+	+

Note: + Sensitive – Resistant

Fig-2: Anti-TB activity for extracts of Ziziphus nummularia ROOT and Standard drugs

1- Aqueous extract

2- Ethanolic extract

3.4. Antituberculosis activity:

The Antituberculosis activity for ethanolic and aqueous extracts of Ziziphus nummularia root was screened against Mycobacterium tuberculosis by Microplate Alamar Blue assay (MABA) method. The MIC was observed in drug concentration which prevents the colour change from blue/black to pink for extracts and standard drug (Table - 4, 5 and Fig 2.

4. CONCLUSION:

Pharmacological evaluation deals with the comparison of aqueous and ethanolic extracts of Ziziphus nummularia root for it’sinvitro antimicrobial and antituberculosis activities. The invitro antimicrobial activity was screened by agar well diffusion method against selected microorganism. The ethanolic extract of Ziziphus nummularia root was more susceptible to Staphylococci with a zone of inhibition 24mm, while aqueous extract showed zone of inhibition 14mm. This study suggested that, ethanolicextract of Ziziphus nummularia root has significant antimicrobial activity, while compared with aqueous extracts. The In vitro antituberculosis activity was screened by Microplate Alamar Blue assay (MABA). The results suggested that aqueousextract of Ziziphus nummularia root has significant antituberculosis activity than ethanolicextracts. It can be concluded that root extracts of Ziziphus nummularia possesses potent antimicrobial and antituberculosis activity. Overall, this approach seems to be one of the best strategies for therapeutic management to overcome bacterial resistance (antimicrobial and antituberculosis). The important outcome of this study will be the development value added product from medicinal plant Ziziphus nummularia for biomedical and herbal based industries.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 09.03.2023 Modified on 29.04.2023

Res. J. Pharmacognosy and Phytochem. 2023; 15(3):198-202.

DOI: 10.52711/0975-4385.2023.00030