INTRODUCTION:

Since time immemorial, plants have continued to be the primary source of medicine used for preventive, curative, or defensive purposes.¹ The plant Ziziphus belongs to the kingdom of lantae, order of rosales, division- magnoliophyta, class- magnoliopsida, family of rhamnaceae and genus- Ziziphus.²This genus is widely distributed in warm temperate and subtropical regions of the world with the highest concentrations occurring in Africa, Asia, and America. They are often thorny shrubs or small trees, typically armed with stipular spines. Modern studies in pharmacognosy and pharmacology have shown that extracts or ingredients isolated from Ziziphus plant exhibit a wide range of pharmacological activities.³

Antimicrobial agents are substances produced by different species of microorganisms like fungi, bacteria, actinomycetes, etc. or any substance which suppress or reduce the growth of other microorganisms and may eventually cause their destruction.⁴Antibiotics were one of the 20th century's greatest human discoveries. It is due to this discovery that there was a major advancement in medical science and numerous patients’ lives have been saved by antibiotics. However, antibiotic resistance is now a significant clinical and public health issue on a global scale.⁵ The major reason for this is inappropriate and random use of antibiotics by common population. Enzymatic degradations, target site alteration, active efflux of drugs are some of the common mechanisms employed by the pathogenic bacteria to produce such resistance.⁶Antibiotics also produce various side effects and adverse reactions due to their indiscriminate use.⁴ This has led to the use of medicinal plants as alternative sources of antibiotics to the synthetic drugs. Crude extracts of herbs serve as sources of antimicrobials and resistance modifying agents because of a wide variety of secondary metabolites present in them. Plant extracts have the ability to bind to protein domains which causes modification or inhibition of protein-protein interactions leading to modulation of host related cellular processes like mitosis, apoptosis, signal transduction and immune response.⁶

Antioxidants are substances which are intimately involved in preventing cellular damage, slowing the ageing process and curing a variety of diseases.⁷ Many plants are natural sources of antioxidants.⁸ These are free-radical scavengers which protect the living organisms from the damage caused by reactive oxygen species.⁹They reduce the effect of dangerous oxidants by binding with these harmful molecules and thereby reducing their destructive power. Anti-oxidation agents also help in repairing the damage sustained by cells.¹⁰

Significant progress has been made in the formulation of novel drug delivery systems (NDDS) for plant actives and extracts over the past several years. Many new herbal formulations containing bioactives and plant extracts have been reported, including polymeric nanoparticles, nanoemulsions, nanocapsules, microspheres, phytosomes, liposomes, ethosomes, and transferosomes. The novel formulations are said to have notable advantages over traditional formulations of plant actives and extracts, including improved bioavailability, enhancement of solubility, protection from physical and chemical degradation, protection from toxicity, enhanced stability, enhanced pharmacological activity, improved tissue macrophage distribution, sustained delivery, and enhanced pharmacological activity.¹¹

Hydrogels are novel drug delivery systems which are very effective in wet wound therapy. They are three-dimensional polymeric networks and have the capability to incorporate a large amount of water along with producing a humid environment and excellent biocompatibility ideal for wound healing.¹² The networks are made up of homopolymers or copolymers and insoluble because they contain chemical or physical crosslinks like crystallites or entanglements.¹³ Hydrogels more closely resemble genuine living tissue compared to other classes of synthetic biomaterials because of their soft consistency, porosity, and high water content.¹⁴ The unique water absorption capacity of hydrogels is attributed to the presence of hydrophilic moieties such as carboxyl and hydroxyl groups and presence of amines, ethers, and sulphates in the polymers.¹⁵

Wound healing is a complicated process and different natural remedies have been used for the healing of wounds. The secondary metabolites present in plants act at different stages of the wound healing process by mechanisms such as angiogenesis, cell proliferation, antioxidant, antimicrobial and anti-inflammatory activities.¹⁶

Now-a-days many of the natural products used in diseases are applied without consideration of proper regulatory aspects, dosage regimens and toxic implications. People also tend to use herbal products as self-medication for various ailments. As a result, study of safety profile of herbal products is necessary.¹⁷ In the present research work dermal toxicity study of Ziziphus oenoplia leaf extract incorporated Carbopol hydrogel formulation is performed to evaluate its safety profile as dermal formulation.

Pharmacological Activities of Ziziphus Plant Extract:

Bukar et al. studied the antibacterial activities of the seed oil extracts of Ziziphus spina-cristi. The antibacterial activities were examined against four different bacteria such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Snigella spp. using agar well diffusion method. The experiments revealed that the Ziziphus spina-cristi seed oil extract showed activities against E.coli, with zone of inhibition of 10mm and Staphylococcus aureus with zone of inhibition of 11mm, which emphasize the fact that the chemical constituents present in the extract like alkaloids, glycosides, tannins, etc. have antibacterial activities on bacterial cell wall and DNA.¹⁸

Aggarwal et al. studied the antioxidant potential of Ziziphus nummularia stem bark hydroalcoholic extract by total antioxidant capacity, DPPH assay, and ferric reducing power. It was observed that the stem bark possessed promising antioxidant activity due to the presence of flavonoids and phenolic contents.¹⁹

Wound healing activity of ethanolic extract of Ziziphus xyloporus Willd. stem bark was examined by Jena et al. The extract showed wound healing activity in excision and incision in-vitro wound models in Swiss Albino rats and promoted angiogenic activity in chorioallantoic membrane model.²⁰

According to a study by Rocchetti et al. Ziziphus lotus plant extract possessed both antimicrobial and prebiotic activities.¹⁵ Dubey et al. studied the antimicrobial activities of saponin, aqueous and methanolic extracts of Ziziphus mauritiana bark against human vaginal pathogens such as P. aeruginosa, E.coli, S. aureus, Klebseilla pneumoniae, Enterobacter faecalis, etc. The extracts significantly inhibited the growth of all the pathogens, saponin extract producing the highest effect.²¹

The antioxidant activity of Ziziphus rugosa Lam. stem bark ethanolic extract was evaluated by Manjunatha et al. The extract showed 81.69% and 55.84% free radical scavenging effect in ABTS and DPPH assays respectively.²²

Abdallah et al. studied the methanolic leaf extract of Ziziphus mauritiana for antimicrobial, antioxidant and anti-inflammatory activities. The study revealed significant antibacterial activities against Bacillus cereus and Proteus vulgaris, and anti-inflammatory properties with 71.83% reduction of inflammation in rats. Promising antioxidant activity was also evaluated.²³Apart from antioxidant property, significant immunomodulatory and cytotoxic activities were also observed in Ziziphus mauritiana dichloromethane extract by Afzal et al.²⁴

Enhanced activities of Ziziphus leaf Extract incorporated formulations:

Antibacterial Activities:

Crystalline silver nanoparticles (AgNPs) of aqueous leaf extract of Ziziphus oenoplia having size of 10nm was synthesized by Soman et al. Antibacterial activity was evaluated using Agar well diffusion method and minimum inhibitory concentration (MIC) was determined against four different bacterial strains of Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae and Escherichia coli. It was observed that the biosynthesized AgNPs possessed much better inhibitory and antibacterial activity in comparison to the aqueous leaf extract and silver nitrate. Bactericidal studies at different time intervals with transmission electron microscopy after AgNPs treatment revealed the presence of AgNPs near cell membrane of bacteria at about 30min exposure and bacterial lysis was observed to be completed in 24 h. This phenomenon provided an insight of the mechanism of bacterial-lysis by direct damage to the cell membrane.²⁵

Asimuddin et al. prepared green synthesized silver nanoparticles using aqueous solution of Ziziphus mauritiana leaves extract as bio-reducing agents. Phytochemical study revealed flavonoid and phenolic contents in the extract. Antibacterial activity of the formulated silver nanoparticles was tested against four different bacterial stains. The nanoparticles inhibited the growth of E. coli moderately with MIC of 5µg/ml and strongly inhibited S. aureus with a MIC of 2.5µg/ml.²⁶ An eco-friendly, simple, rapid, and cost-effective method was used by Abdelkar et al. to prepare green synthesized AgNPs as an antimicrobial agent using Ziziphus spina-christi leaf extract. The extract could reduce silver ions into silver by stirring for 30mins with magnetic stirrer. Antibacterial properties of AgNPs were found to be active against common human pathogens of K. pneumoniae, A. baumanni, E. coli and P. aeruginosa bacterial stains.²⁷

Antioxidant activities:

Ziziphus nummularia leaf extract gold nanoparticles were formulated and evaluated for antioxidant activity by Padalia H et al. The results revealed that the photosynthesized AuNPs possessed effective dose-dependent in-vitro antioxidant activity against, DPPH (IC₅₀ = 520μg/ml), ABTS (IC₅₀ = 690μg/ml) and SO (IC₅₀ = 330μg/ml) radicals.²⁸

Other Formulations Using Ziziphus Herbal Extract:

Sobhani et al. formulated Ziziphus jujuba Mill fruit syrup and evaluated its physicochemical properties in accelerated stability conditions which was considered as a potential medicine for large scale industrial production.²⁹A stable water in oil emulsion containing Ziziphus mauritiana hydroalcoholic leaf extract was successfully formulated by Akhtar et al. for skin rejuvenation activity.³⁰Mucoadhesive discs containing Ziziphus jujuba stem bark extract was formulated by Hamedi S et al. for curing buccal diseases.³¹A hair tonic possessing hair growth promoting activity was also formulated by Sekhar et al. using Ziziphus jujuba leaf extract which produced comparable hair growth rate with 2% Minoxidil.^32,33

Ziziphus leaf extract incorporated hydrogel formulation:

In this work a hydrogel formulation has been prepared by adding 1g Carbomer 934 to 100ml distilled water and stirring for 4 hours. Triethanolamine was added with further stirring till the pH was neutral. 1% ethanolic leaf extract of Ziziphus oenoplia (previously evaluated for effective antibacterial and antioxidant activity) and 0.1% methyl paraben as preservative was added and the formulation was stored at 4ᵒC.³⁴ The pH of the formulation was determined just after the preparation and after 30 days, 60 days and 90 days with the help of a digital pH meter by dipping the electrode completely into the formulation. In all the conditions the pH of the hydrogel was found to be nearly constant ensuring the stability of the formulation.³⁵

Further, the safety profile of the hydrogel formulation was observed by conducting dermal toxicity study on adult male Albino Wister rats. The animal experiments were carried out in accordance with the guidelines of the Institutional Animal Ethics Committee (registration no. 1938/PO/Rc/S/17/CPCSEA) following the OECD Principles of Good Laboratory Practices, Schedule Y requirements of Drugs and Cosmetics Act. The test item was applied topically on the shaved area of the dorsolateral trunk of the rats. 20 rats were divided into four dose groups – negative control, hydrogel with extract low (1g/kg), middle (2g/kg), and high (4g/kg) doses, each group containing 5 rats. 0.9% NaCl solution was taken as control. The test items were uniformly applied on the shaved areas of the rat skin once a day over a period of 28 days. The animals were observed at time intervals of 0, 7, 14, 21 and 28 days in order to evaluate the skin reaction (such as erythema, edema and necrosis), food consumption and variations in body weight. The animals were also observed once daily for clinical signs and twice daily for mortality or morbidity throughout the experiment.

At the end of the animal study all the groups of rats showed no clinical signs of intoxication, no skin reactions, mortality, or morbidity. No drastic change in food consumption or body weight were observed on the test and the control groups of rats. This study shows that the Carbopol hydrogel formulation containing leaf extract of Ziziphus oenoplia is safe to be applied on the skin. Further evaluation of wound healing potential of the formulated hydrogel will be conducted in future.

FUTURE PROSPECTS:

From this study it can be observed that herbal extracts of the genus Ziziphus can be extensively used for different ailments as alternatives to synthetic drugs, with improved properties and less toxicity. For effective drug administration different types of dosage forms can be formulated with the herbal extracts of this plant. Here, a hydrogel formulation containing Ziziphus oenoplia leaf extract has been prepared and dermal toxicity study has been conducted on rat models. Further evaluation of wound healing of the hydrogel formulation is the future prospect of the work.

CONCLUSION:

For many years natural medicine has been the basis of remedy for various illnesses.³⁶ According to a survey, more than 80% of the world’s population depends on traditional or herbal medicine for their basic ailments.³⁷In this study it is found that the various plant parts of Ziziphus such as root bark, leaves, stem bark, seed oil, root, stem, etc. and their different formulations possess a wide variety of pharmacological activities such as wound healing, antibacterial, antioxidant, antiinflammatory, immunomodulatory, etc. due to the presence of different secondary metabolites in the plant extracts. It is also observed that the formulated Carbopol hydrogel containing Ziziphus oenoplia leaf extract presented no clinical signs of toxicity, mortality/morbidity, skin reactions or any notable change in food consumption or body weight in the rat models when applied dermally on the test and the control groups of male Albino Wister rats. Thus, from this study it can be inferred that the Carbopol hydrogel formulation containing herbal extract of Ziziphus oenoplia is safe for application on the skin. The antibacterial and antioxidant properties of the leaf extract will aid in the wound healing activity of the herbal hydrogel.

REFERENCES:

1. Sukmandari NS, Dash GK, Jusof WWH, Hanafi M, A Review on Nephelium lappaceum L. Res J Pharm Technol. 2007. 10(8), 1-9. DOI: 10.5958/0974-360X.2017.00498.X

2. Raghvendra D, Dubey K, Sridhar C, Jayaveera KN, Human vaginal pathogen inhibition studies on aqueous, methanolic and saponins extracts of stem barks of Ziziphus mauritiana. Int.J. Pharm. Sci. Res. 2011. 2(3), 659-663.DOI: 10.13040/IJPSR.0975-8232.2(3)

3. Maaiden EE, Kharrassi YE, Qarah NAS, Essamadi AK, Moustaid K, Nasser B, Genus Ziziphus: A Comprehensive review on ethnopharmacological, phytochemical and pharmacological properties, J. Ethnopharmacol. 2019. S0378-8741(19), 33861-861. DOI: https://doi.org/10.1016/j.jep.2020.112950

4. Soares GMS, Figueiredo LC, Faveri M, Cortelli SC, Duarte PM, Feres M, Mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs. J Appl Oral Sci. 2012. 20(3), 295-309. DOI: 10.1590/s1678-77572012000300002

5. Sreeja MK, Gowrishankar NL, Adisha S, Divya KC, Antibiotic resistance-reasons and the most common resistant pathogens - A review. Res J Pharm Technol. 2017. 10(6), 1886 -90. DOI: 10.5958/0974-360X.2017.00331.6

6. Gupta PD, Birdi TJ, Development of botanicals to combat antibiotic resistance. J Ayurveda Integr Med. 2017. 8(4), 266-75. DOI: 10.1016/j.jaim.2017.05.004

7. Prithviraj C, Suresh K, Debarupa D , Gupta Vikas, Role of Antioxidants in Common Health Diseases. Res J Pharm Technol. 2009. 2(2), 234-44.

8. Khe JC, Shivkanya F, Vetriselvan S, Mahendran S, Kathiresan V. S, Saminathan K, Seng WY, Raju VVSS, Kumar JA, Hasan MMN, Kumar FN, Phytochemical screening and antioxidant activity of cananga odorata extract. Res J Pharm Technol. 2022. 15(3), 1230-34. DOI : 10.52711/0974-360X.2022.00205

9. Pallab K, Satyendra D, Kanta SR,. A review on ethanolic plants of Assam (India) and their Anti-oxidant activity. J. Med Pharm Allied Sci. 2014. 6(3), 20-28.

10. Mathew S, Abraham TE, Studies on the antioxidant activities of cinnamon (Cinnamomum verum) bark extracts, through various in vitro models. Food Chem. 2006. 94(2006), 520-28. DOI: 10.1016/j.foodchem.2004.11.043

11. Saraf AS, Applications of novel drug delivery system for herbal formulations. Fitoterapia. 2010. 81 (2010), 680–689. DOI: 10.1016/j.fitote.2010.05.001

12. Gavan A, Colobatiu L, Hanganu D, Bogdan C, Olah NK, Achim M, Mirel S, Development and Evaluation of Hydrogel Wound Dressings Loaded with Herbal Extracts. Processes. 2022. 10(242): 1-12. DOI: org/10.3390/pr10020242

13. Sawant SV, Sankpal SV, Jadhav KR, Kadam VJ. Hydrogel as drug delivery system. Res J Pharm Technol. 2012, 5(5), 561-569.

14. Gharat JS, Dalvi YV, Compressive Review on Hydrogel. Res. J. Pharm Technol. 2018. 8(3), 172-181. DOI: 10.5958/2231- 5713.2018.00028.4

15. Fathi M, Ahmadi N, Forouhar A, Atani SH. Natural Hydrogels, the interesting carriers for herbal extracts. Food Rev. Int. 2021. Feb,1-25. DOI: .org/10.1080/87559129.2021.1885436

16. Vitale S, Colanero S, Placidi M, Emidio GD, Tatone C, Amicarelli F, D’Alessandro AM, Phytochemistry and biological activity of medicinal plants in wound healing: An overview of current research. Molecules. 2022. 27(3566), 1-30. DOI: 10.3390/molecules27113566

17. Mishra AK, Mishra A, Chattopadhyay P. Screening of acute and sub-chronic dermal toxicity of Calendula officinalis L essential oil. Regul. Toxicol. Pharmacol. 2018, 98(2018). 184-189. DOI: 10.1016/j.yrtph.2018.07.027

18. Bukar AM, Kyari MZ, Gwaski PA, Gudusu M, Kuburi FS, Abadam YI, Evaluation of phytochemical and potential antibacterial activity of Ziziphus spinachristi L. against some medically important pathogenic bacteria obtained from University of Maiduguri Teaching Hospital, Maiduguri, Borno State - Nigeria. J. pharmacogn. Phytochem. 2015. 3(5), 98-101.

19. Aggarwal B, Sharma P, Lamba HS, Assessment of Phytochemical and in vitroAntioxidant Potential of Zizyphus nummularia Stem Bark. Int J Pharma Res Health Sci. 2018. 6(2), 2502-06. DOI:10.21276/ijprhs.2018. 2018. 02.29

20. Jena BK, Ratha B, Kar S, Mohanta S,Tripathy M, Nayak AK, Wound healing potential of Ziziphus xylopyrus WILLD. (Rhamnaceae) stem bark ethanol extract using in vitro and in vivo model. J. Drug Deliv.Ther. 2012. 2(6), 41-46.

21. Rocchetti G, Alcántara C, Bäuerl C, García-Pérez JV, José, Lorenzo JM, Lucini L, Collado MC, Barba FJ, Bacterial growth and biological properties of Cymbopogon schoenanthus and Ziziphus lotus are modulated by extractionconditions. Food Res. 2020. 136(109534), 1-10. DOI: 10.1016/j.foodres.2020.109534

22. Manjunatha E, Vedigounder M, Geetha KM, Nandeesh R, Ahmed SM, Antioxidant and Antiulcer activity of Ziziphus rugosa Lam. Bark. Res J Pharm. Technol. 2020. 13(10), 4720-4724. DOI: 10.5958/0974-360X.2020.00831.8

23. Abdallah EM, Elsharkawy ER, Ed-dra A, Biological activities of\methanolic leaf extract of Ziziphus mauritiana. Biosci. Biotech. Res. Comm. 2016. 9(4), 605-14.

24. Afzal S, Batool M, Ahmad Ch BA, Ahmad A, Uzair M,Afzal K, Immunomodulatory, Cytotoxicity, and AntioxidantActivities of Roots of Ziziphus mauritiana,. Pharmacogn. Mag. 2017. 13 (50), S262–S265. DOI: 10.4103/pm.pm_398_16

25. Soman S, Ray JG, Silver nanoparticles synthesized using aqueous leaf extract of Ziziphus oenoplia (L.) Mill: Characterization and assessment of antibacterial activity. J. Photochem. Photobiol. B, Biol. 20016. 163(2016), 391-402. DOI: 10.1016/j.jphotobiol.2016.08.033

26. Asimuddin M, Shaik M R, Fathima N, Afreen MS, Adil SF, Siddiqui MRH, Jamil K, Khan M, Study of Antibacterial Properties of Ziziphus mauritiana based Green Synthesized Silver Nanoparticles against Various Bacterial Stains. Sustainability. 2020. 12(1484), 1-14. DOI: 10.3390/su12041484

27. Amdelkar H, Alzahrani H, Al-Ayafi A, Al-Mulah H, Al-Zubaidi S, Green Synthesis, Characterization and Antimicrobial Activity of Biosynthesized Silver Nanoparticles using Ziziphus spina- cristi Leaf Extracrs. HSOA Adv in MicrobioResearch., 2019. 3(010), 1-7. DOI: 10.24966/AMR-694X/100010

28. Padalia H, Chanda S, Antioxidant and Anticancer Activities of Gold Nanoparticles Synthesized Using Aqueous Leaf Extract of Ziziphus nummularia. BioNanoScience. 2021, 281-294. DOI:10.1007/s12668-021-00849-y

29. Sobhani Z, Emami SA, Bazzaz BSF, Rajabi O, A modern formulation of traditional medicine: Jujube (Ziziphus jujuba Mill.) fruit syrup. Indian J. Tradit. Knowl. 2019. 18(4),662-669.

30. Akhtar N, Ijaz S, Khan HMS, Uzair B, Reich A, Khan BA, Ziziphus mauritiana leaf extract emulsion for skin rejuvenation. Trop. J. Pharm. Res. 2016. 15(5), 929-936. DOI: 10.4314/tjpr.v15i5.5

31. Hamedi S, Shams-Ardakani MR, Sadeghpour O, Amin G, Hajighasemali D, Orafai H, Designing mucoadhesive discs containing stem bark extract of Ziziphus jujuba based on Iranian traditional documents. Iran J Basic Med Sci. 2016. 19(3), 330-336.

32. Sekar M, Priya NV, Ramesh R, Kumar SG, Bharati AD, Afsha SA, Formulation and Evaluation of Hair Tonic from Ziziphus Jujuba Leaf Extract. Int. j. pharm. chem. biol. sci. 2016. 6(4), 447-454.

33. Ali Y, Nada MR, Hameeda AN, Taghreed F, Salwa H, Inaam J, Furqan S, Ban S, Allelopathi Effect of the Leaf Extracts of Zizyphus spina-christi (Sedr), Salix fragilis L. and Ricinus communis L. on Seed Germination Percentage of Triticumaestivum L. in Iraq. Research J. Pharm. and Tech. 2018.11(1), 187-192. DOI: 10.5958/0974-360X.2018.00035.5

34. Chirayath RB, Viswanathan AA, Jayakumar R, Biswas R, Vijayachandran LS, Development of Mangifera indica leaf extract incorporated carbopol hydrogel and its antibacterial efficacy against Staphylococcus aureus. Colloids Surf. B. 2019. 178(2019), 377–384. DOI: 10.1016/j.colsurfb.2019.03.034

35. Haneefa KP M, Hanan K S, Saraswathi R, Mohanta GP, Nayar C, Formulation and evaluation of herbal gel of Pothos scandens Linn. Asian Pac. J. Trop. Med. 2010. Dec(2010), 988-992. DOI:10.1016/S1995-7645(11)60015-1

36. Baidya M, Jayaraman A, Maji HS, Krishna P S R, Das D, Evaluation of acute and sub-acute toxicity of Sivanar amirtham in albino mice and wistar albino rats. J. med. pharm. allied sci. 2022. Jan-Feb; 11(1), 2217, 4196-4204. DOI:10.22270/jmpas.V11I1.2217

37. Baidya M, Maji HS, Bhatt S, Das D. In-vitro evaluation of the thrombolytic and anti-inflammatory activity of Capparis sepiaria root extracts. J. med. pharm. allied sci. 2022. Jan-Feb; 11(1), 1879, 4166-4171. DOI:10.22270/jmpas.V11I1.1879

Received on 13.02.2022 Modified on 17.03.2022

Research J. Pharm. and Tech 2023; 16(7):3300-3304.

DOI: 10.52711/0974-360X.2023.00544