Phytochemical screening, antimicrobial activity, in vitro and in vivo antioxidant activity of Berberis lycium Royle root bark extract

Triagem fitoquímica, atividade antimicrobiana, atividade antioxidante in vitro e in vivo do extrato de casca de raiz de Berberis lycium Royle

T. A. Mughal S. Ali A. Hassan S. A. R. Kazmi M. Z. Saleem H. A. Shakir S. Nazer M. A. Farooq M. Z. Awan M. A. Khan S. Andleeb S. Mumtaz S. Mumtaz H. M. Tahir N. Gulzar About the authors

Abstract

Antioxidants are materials that scavenge or remove free radicals from living systems. The oxidation process ends in the production of free radicals. These free radicals are the chief birthplace of cancerous cells. Antioxidizing agents remove free radical intermediates by terminating oxidation processes by being oxidized themselves. On the other hand, infectious diseases affect the world on a large scale. To fight these diseases several synthetic compounds have been used. Plant based medications play important role in this regard. So, the current research aimed to investigate the antibacterial and antioxidant effect of Berberis lycium Royle root bark (BLR) extract. Berberis lycium Royle was used for phytochemical analysis and also as antimicrobial and antioxidant agents. The antimicrobial activity was evaluated by the agar well diffusion method. Current study revealed that BLR was rich in phytochemicals and toxic against tested pathogenic bacteria. BLR showed the highest activity against S. pyogenes (13.3±0.8 mm). The lowest antibacterial activity was reported against E. coli (0±0 mm). In case of minimum inhibitory concentration, it was observed that BLR with 10 μg/mL concentration showed the highest activity while 2.5 μg/mL of BLR showed the least inhibitory activity. The highest In vitro antioxidant activity was recorded as 65% at 100 µg/mL. In case of in vivo antioxidant activity level of CAT, GSH and SOD were decreased while that of MDA was enhanced in groups treated with CCl4 as compared to the control group. BLR extract treatment reversed all these changes significantly. Current results indicate that BLR is effective against bacterial pathogens and also has antioxidant potential.

Keywords:
Berberis lycium Royle; root bark extract; phytochemicals; antibacterial; antioxidant

Resumo

Os antioxidantes são materiais que eliminam ou removem os radicais livres dos sistemas vivos. O processo de oxidação termina na produção de radicais livres. Esses radicais livres são o principal local de nascimento das células cancerosas. Os agentes antioxidantes removem os intermediários dos radicais livres ao encerrar os processos de oxidação ao serem eles próprios oxidados. Por outro lado, as doenças infecciosas afetam o mundo em grande escala. Para combater essas doenças, diversos compostos sintéticos têm sido utilizados. Os medicamentos à base de plantas desempenham um papel importante a este respeito. Assim, o objetivo da pesquisa atual é investigar o efeito antibacteriano e antioxidante do extrato da casca da raiz de Berberis lycium Royle (BLR). Berberis lycium Royle foi utilizado para análises fitoquímicas e também como agentes antimicrobianos e antioxidantes. A atividade antimicrobiana foi avaliada pelo método de difusão em ágar em poço. A partir do estudo atual, observou-se que o BLR era rico em fitoquímicos e tóxico contra bactérias patogênicas testadas. BLR apresentou maior atividade contra S. pyogenes (13,3 ± 0,8 mm). A menor atividade antibacteriana foi relatada contra E. coli (0 ± 0 mm). No caso de concentração inibitória mínima, observou-se que BLR com concentração de 10 μg / mL apresentou maior atividade, enquanto BLR 2,5 μg / mL apresentou menor atividade inibitória. A maior atividade antioxidante in vitro foi registrada como 65% a 100 µg / mL. No caso do nível de atividade antioxidante in vivo de CAT, GSH e SOD diminuiu, enquanto o de MDA aumentou nos grupos tratados com CCl4 em comparação com o grupo controle. O tratamento com extrato de BLR reverteu todas essas mudanças significativamente. Os resultados atuais indicam que o BLR é eficaz contra patógenos bacterianos e também tem atividade antioxidante.

Palavras-chave:
Berberis lycium Royle; extrato da casca da raiz; fitoquímicos; antibacteriano; antioxidante

1. Introduction

Bacterial infections cause millions of infection-based morbidities and mortalities annually and became the worldwide open health matter (Ji et al., 2016JI, Y., THOMAS, C., TULIN, N., LODHI, N., BOAMAH, E., KOLENKO, V. and TULIN, A.V., 2016. Charon mediates immune deficiency-driven PARP-1-dependent immune responses in Drosophila. Journal of Immunology, vol. 197, no. 6, pp. 2382-2389. http://dx.doi.org/10.4049/jimmunol.1600994. PMid:27527593.
http://dx.doi.org/10.4049/jimmunol.16009...
). Microorganisms are cosmopolitan and cause various types of infectious diseases, hence they are termed pathogens. Every microbe has a different potential for pathogenicity. The efficacy of available antibiotics is endangered by pathogen resistance (Tong et al., 2018TONG, C., ZOU, W., NING, W., FAN, J., LI, L., LIU, B. and LIU, X., 2018. Synthesis of DNA-guided silver nanoparticles on a graphene oxide surface: enhancing the antibacterial effect and the wound healing activity. RSC Advances, vol. 8, no. 49, pp. 28238-28248. http://dx.doi.org/10.1039/C8RA04933E.
http://dx.doi.org/10.1039/C8RA04933E...
) which is a global health problem today. This peril is increasing day by day and breeds the requirement for innovative antibiotics with value-added bactericidal activities. Phyto-based medications are the need of the hour because of their eco-friendly nature. Medicinal plants are potential source of antibacterial drugs. Many of the synthetic drugs were discovered either directly or indirectly from the plant source (Swaroopa et al., 2017SWAROOPA, P., REDDY, V.J.S., KOSHMA, M., SUDHARANI, Y., BASHA, S.J. and ADITHYA, T.N., 2017. Review on anti-diabetic activity on medicinal plants. International Journal of Pharmacology, vol. 7, no. 12, pp. 230-235.).

Antioxidants are, the substances that deal with the free radicals. Free radicals are the cause of numerous diseases like atherosclerosis, cancer, diabetes and liver cirrhosis (Slatern, 1987). Antioxidants involve in protecting the human body against the damage caused by free radicals. The antioxidants play their role as reducing agents, being oxidized themselves (Sies, 1997SIES, H., 1997. Oxidative stress: oxidants and antioxidants. Experimental Physiology, vol. 82, no. 2, pp. 291-295. http://dx.doi.org/10.1113/expphysiol.1997.sp004024. PMid:9129943.
http://dx.doi.org/10.1113/expphysiol.199...
). Antioxidants, natural and artificial, have hydroxyl groups involved in the redox reactions. Antioxidants that occur naturally include vitamins (B, C, E), phytochemicals (mostly flavonoids) and some minerals having cupper, iron, zinc, manganese and selenium metals (Hamid et al., 2010HAMID, A.A., AIYELAAGBE, O.O., USMAN, L.A., AMEEN, O.M. and LAWAL, A., 2010. Antioxidants: its medicinal and pharmacological applications. African Journal of Pure and Applied Chemistry, vol. 4, pp. 142-151.). Phenolic compounds involved in the capturing and scavenging of free radicals act as synthetic antioxidants (Ansari et al., 2021ANSARI, S.S., DIÑO, P.H., CASTILLO, A.L. and SANTIAGO, L.A., 2021. Antioxidant activity, xanthine oxidase inhibition and acute oral toxicity of Dillenia philippinensis Rolfe (Dilleniaceae) leaf extract. Journal of Pharmacy & Pharmacognosy Research, vol. 9, no. 6, pp. 846-858.).

Berberis lycium Royle (family: Berberidaceae), a plant, called barberry in English, the word “kashmal” and “Darhald” are used for fruit and roots respectively. B. lycium Royle is usually present in the Himalayas area (Kaur and Miani, 2001KAUR, C. and MIANI, S.B., 2001. Fruits and vegetables health foods for new millennium. Indian Journal of Horticulture, vol. 45, no. 4, pp. 29-32.). Intestinal colic, diarrhea, jaundice, piles, ophthalmia, internal wounds, rheumatoid arthritis, gingivitis, diabetes, back pain, throat pain, scabies, pustules, remittent fever, broken bones, sun blindness, menorrhagia and intermittent fever are treated by using B. lycium Royle. B. lycium Royle is also playing its role as a diaphoretic, diuretic, febrifuge, and expectorant (Shabbir et al., 2012SHABBIR, A., SHAHZAD, M., ARFAT, Y., ALI, L., AZIZ, R.S., MURTAZA, G. and WAQAR, S.A., 2012. Berberis lyceum Royle: A review of its traditional uses, phytochemistry and pharmacology. African Journal of Pharmacy and Pharmacology, vol. 6, no. 31, pp. 2346-2353.).

In many traditional preparations, different parts of this plant have been used. Dry powder of bark is used to treat throat pain, dysentery and wounds. Root bark aqueous extract is used for the treatment of diabetes, scabies and pustules. Root bark powdered paste has been used in the treatment of bone fracture, gingivitis and rheumatism (Ahmed et al., 2004AHMED, E., ARSHAD, M., AHMAD, M., SAEED, M. and ISHAQUE, M., 2004. Ethno-pharmacological survey of some medicinally important plants of Galliyat areas of NWFP, Pakistan. Asian Journal of Plant Sciences, vol. 3, no. 4, pp. 410-415. http://dx.doi.org/10.3923/ajps.2004.410.415.
http://dx.doi.org/10.3923/ajps.2004.410....
). Traditional medicine systems involved in the development of modern medicines for curing numerous diseases.

According to various studies, many newly known targets of the drug are moderated by plant components. Traditional drugs should be renewed concerning the recent understanding of modern or allopathic medications. B. lycium Royle has been used as an anti-diabetic medicine for various times. Recent research work has mounted that B. lyceum Royle act as insulin, involves in reducing hyperglycemia. The major alkaloid in B. lycium Royle, Berberine, involved in the anti-inflammatory effect. To treat wounds, B. lycium Royle is suggested by local consultants. B. lycium Royle plays its role in healing wounds by increasing the deposition of collagen and epithelialization area. For the treatment of different diseases, new drug targets and modern medicines are being developed from the knowledge of traditional medicines (Aggarwal et al., 2006AGGARWAL, B.B., ICHIKAWA, H., GARODIA, P., WEERASINGHE, P., SETHI, G., BHATT, I.D., PANDEY, M.K., SHISHODIA, S. and NAIR, M.G., 2006. From traditional ayurvedic medicine to modern medicine: identification of therapeutic targets for suppression of inflammation and cancer. Expert Opinion on Therapeutic Targets, vol. 10 no. 1, pp. 87-118. http://dx.doi.org/10.1517/14728222.10.1.87. PMid:16441231.
http://dx.doi.org/10.1517/14728222.10.1....
).

The current study aimed to find out the alternatives of the already available antibacterial and antioxidant agents. The extracts of B. lycium Royle were prepared and analyzed for antibacterial and antioxidant activity.

2. Materials and Methods

2.1. Ethical statement

Animal trials were carried out in accord with indigenous (law of Government College University, Lahore, Pakistan) and international law (Wet op de dierproeven, Wod, Article 9 of Dutch Law as mentioned in our former studies (Dar et al., 2019DAR, K.K., ALI, S., EJAZ, M., NASREEN, S., ASHRAF, N., GILLANI, S.F., SHAFI, N., SAFEER, S., KHAN, M.A., ANDLEEB, S. and MUGHAL, T.A., 2019. In vivo induction of hepatocellular carcinoma by diethylnitrosoamine and pharmacological intervention in Balb C mice using Bergenia ciliata extracts. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 79, no. 4, pp. 629-638. http://dx.doi.org/10.1590/1519-6984.186565. PMid:31017181.
http://dx.doi.org/10.1590/1519-6984.1865...
; Mughal et al., 2019MUGHAL, T.A., SALEEM, M.Z., ALI, S., ANWAR, K.K., BASHIR, M.M., BABAR, M. and KHAN, M.A., 2019. Evaluation of hepatotoxicity of carbon tetrachloride and pharmacological intervention by vitamin E in Balb C mice. Pakistan Journal of Zoology, vol. 51, no. 2, pp. 755-761. http://dx.doi.org/10.17582/journal.pjz/2019.51.2.755.761.
http://dx.doi.org/10.17582/journal.pjz/2...
; Mumtaz et al., 2019MUMTAZ, S., ALI, S., KHAN, R., ANDLEEB, S., ULHAQ, M., KHAN, M.A. and SHAKIR, H.A., 2019. The protective role of ascorbic acid in the hepatotoxicity of cadmium and mercury in rabbits. Environmental Science and Pollution Research International, vol. 26, no. 14, pp. 14087-14096. http://dx.doi.org/10.1007/s11356-019-04620-5. PMid:30852747.
http://dx.doi.org/10.1007/s11356-019-046...
; Ali et al., 2020ALI, S., EJAZ, M., DAR, K.K., NASREEN, S., ASHRAF, N., GILLANI, S.F., SHAFI, N., SAFEER, S., KHAN, M.A., ANDLEEB, S., AKHTAR, N. and MUGHAL, T.A., 2020. Evaluation of chemopreventive and chemotherapeutic effect of Artemisia vulgaris extract against diethylnitrosamine induced hepatocellular carcinogenesis in Balb C mice. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 80, pp. 489-496. PMid:31691741.; Mughal and Ali, 2020MUGHAL, T.A. and ALI, S., 2020. Assessment of antidiabetic potential of Berberis lycium Royle root bark extract in experimental animal model. Authorea. In press.; Mughal et al., 2020aMUGHAL, T.A., ALI, S., HASSAN, A., SALEEM, M.Z., MUMTAZ, S. and MUMTAZ, S., 2020a. Carbon tetrachloride-induced hepatocellular damage in Balb C mice and pharmacological intervention by extract of Daucus carota. RADS Journal of Pharmacy and Pharmaceutical Sciences, vol. 8, no. 4, pp. 1-9.; Mughal et al., 2020bMUGHAL, T.A., ALI, S., TAHIR, H.M., MUMTAZ, S., MUMTAZ, S., HASSAN, A. and KAZMI, S.A.R., 2020b. Anti-hyperlipidemic effect of Berberis lycium Royle root bark extract in alloxanized Swiss albino mice. Punjab University Journal of Zoology, vol. 35, no. 2, pp. 261-268. http://dx.doi.org/10.17582/journal.pujz/2020.35.2.261.268.
http://dx.doi.org/10.17582/journal.pujz/...
).

2.2. Collection of medicinal plant

Berberis lycium Royle plant was collected from Battangi Mughalan, Chinari, District Jhelum valley, Azad Kashmir (Figure 1). The plant was recognized by ethno-phytologist, Department of Botany, University of Azad Jammu and Kashmir (UAJK) Muzaffarabad. The plant was washed with tap water to confiscate dust. Shade dried plant parts were creased into fine powder.

Figure 1
(A): Berberis lycium Royle, (B): Map of Pakistan. Arrow indicates the localization of plant collection.

2.3. Preparation of plant extract

Plant powder (10 g) was boiled with distilled water (100 mL). The extract was filtered using Whatman No. 1 filter paper and intended by a rotary evaporator (R-200 Buchi, Switzerland). Plants extract was dried in a vacuum oven (Vacucell, Einrichtungen GmbH) at 40 °C (Figure 2) as mentioned in our previous studies (Mughal et al., 2020aMUGHAL, T.A., ALI, S., HASSAN, A., SALEEM, M.Z., MUMTAZ, S. and MUMTAZ, S., 2020a. Carbon tetrachloride-induced hepatocellular damage in Balb C mice and pharmacological intervention by extract of Daucus carota. RADS Journal of Pharmacy and Pharmaceutical Sciences, vol. 8, no. 4, pp. 1-9.; Mughal et al., 2020bMUGHAL, T.A., ALI, S., TAHIR, H.M., MUMTAZ, S., MUMTAZ, S., HASSAN, A. and KAZMI, S.A.R., 2020b. Anti-hyperlipidemic effect of Berberis lycium Royle root bark extract in alloxanized Swiss albino mice. Punjab University Journal of Zoology, vol. 35, no. 2, pp. 261-268. http://dx.doi.org/10.17582/journal.pujz/2020.35.2.261.268.
http://dx.doi.org/10.17582/journal.pujz/...
; Mughal and Ali, 2020MUGHAL, T.A. and ALI, S., 2020. Assessment of antidiabetic potential of Berberis lycium Royle root bark extract in experimental animal model. Authorea. In press.).

Figure 2
Schematic illustration of research plan.

2.4. Phytochemical screening

The aqueous extract of BLR root bark was tested for the presence of alkaloids, steroids, tannins, saponins, glycosides, terpenoids, proteins, free amino acids, carbohydrates, phenols and flavonoids according to a method mentioned in Souza et al. (2020)SOUZA, A.O., BESSA, D.H.R.F., FERNANDES, C.C., PEREIRA, P.S., MARTINS, C.H.G. and MIRANDA, M.L.D., 2020. Phytochemical screening of extracts from Spiranthera odoratissima A. St.-Hil. (Rutaceae) leaves and their in vitro antioxidant and anti-Listeria monocytogenes activities. Acta Scientiarum. Biological Sciences, vol. 42, no. 1, p. e51881. http://dx.doi.org/10.4025/actascibiolsci.v42i1.51881.
http://dx.doi.org/10.4025/actascibiolsci...
as shown in Table 1.

Table 1
The test for different phytochemical constituents.

2.5. Antibacterial activity

2.5.1. Pathogenic bacteria tested

Seven clinical pathogens such as Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Serratia marcescens (Gram-negative bacteria) Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus pyogenes (Gram-positive Bacteria) were used.

2.5.2. Agar well diffusion assay

The antimicrobial activity was evaluated by the agar well diffusion method (Valgas et al., 2007VALGAS, C., SOUZA, S.M., SMÂNIA, E.F.A. and SMÂNIA JÚNIOR, A., 2007. Methods to determine antibacterial activity of natural products. Brazilian Journal of Microbiology, vol. 38, no. 2, pp. 369-380. http://dx.doi.org/10.1590/S1517-83822007000200034.
http://dx.doi.org/10.1590/S1517-83822007...
).

2.5.3. Minimum Inhibitory Concentration (MIC)

The bactericidal activity of BLR was experienced using the standard microdilution method, which concludes the minimum inhibitory concentration (MIC) leading to the inhibition of microbial growth. MIC of BLR extract was determined by the agar well diffusion method. Different concentrations of extract (2.5 μg, 5 μg and 10 μg) per 1 ml of DMSO were used for antimicrobial activity. The least concentration that did not show growth of tested microorganisms was considered as MIC (Eloff, 1998ELOFF, J.N., 1998. A sensitive and quick micro plate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica, vol. 64, no. 8, pp. 711-713. http://dx.doi.org/10.1055/s-2006-957563. PMid:9933989.
http://dx.doi.org/10.1055/s-2006-957563...
).

2.6. In vitro antioxidant activity

2.6.1. DPPH radical-scavenging activity

The radical scavenging activity of the BLR extract was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH (Kwon et al., 2007KWON, Y.I., APOSTOLIDIS, E., KIM, Y.C. and SHETTY, K., 2007. Health benefits of traditional corn, beans, and pumpkin: in vitro studies for hyperglycemia and hypertension management. Journal of medicinal food, vol. 10, no. 2, pp. 266-275.). 1.0 ml of the prepared DPPH (0.1 mM in ethanol) was added to different concentrations (0.5, 1 and 1.5 ml) of BLR-Extract. Reaction mixture was shaken and then incubated for 30 minutes in the darkness. The absorbance was recorded at 517 nm after thirty minutes. For the positive control, ascorbic acid was used. Higher radical scavenging activity was the indication of lower absorbance. Radical scavenging activity was expressed as the inhibition percentage of free radical by the sample and was calculated using the subsequent formula (Equation 1):

% inhibition = Absorbance of control Absorbance of test Absorbance of control × 100 (1)

2.7. In vivo antioxidant activity

In vivo antioxidant activity was studied by using mice as the experimental model.

2.8. Animal management

Animals were managed according to the method discussed in different studies (Dar et al., 2019DAR, K.K., ALI, S., EJAZ, M., NASREEN, S., ASHRAF, N., GILLANI, S.F., SHAFI, N., SAFEER, S., KHAN, M.A., ANDLEEB, S. and MUGHAL, T.A., 2019. In vivo induction of hepatocellular carcinoma by diethylnitrosoamine and pharmacological intervention in Balb C mice using Bergenia ciliata extracts. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 79, no. 4, pp. 629-638. http://dx.doi.org/10.1590/1519-6984.186565. PMid:31017181.
http://dx.doi.org/10.1590/1519-6984.1865...
; Mughal et al., 2019MUGHAL, T.A., SALEEM, M.Z., ALI, S., ANWAR, K.K., BASHIR, M.M., BABAR, M. and KHAN, M.A., 2019. Evaluation of hepatotoxicity of carbon tetrachloride and pharmacological intervention by vitamin E in Balb C mice. Pakistan Journal of Zoology, vol. 51, no. 2, pp. 755-761. http://dx.doi.org/10.17582/journal.pjz/2019.51.2.755.761.
http://dx.doi.org/10.17582/journal.pjz/2...
; Ali et al., 2020ALI, S., EJAZ, M., DAR, K.K., NASREEN, S., ASHRAF, N., GILLANI, S.F., SHAFI, N., SAFEER, S., KHAN, M.A., ANDLEEB, S., AKHTAR, N. and MUGHAL, T.A., 2020. Evaluation of chemopreventive and chemotherapeutic effect of Artemisia vulgaris extract against diethylnitrosamine induced hepatocellular carcinogenesis in Balb C mice. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 80, pp. 489-496. PMid:31691741.; Mughal and Ali, 2020MUGHAL, T.A. and ALI, S., 2020. Assessment of antidiabetic potential of Berberis lycium Royle root bark extract in experimental animal model. Authorea. In press.; Mughal et al., 2020aMUGHAL, T.A., ALI, S., HASSAN, A., SALEEM, M.Z., MUMTAZ, S. and MUMTAZ, S., 2020a. Carbon tetrachloride-induced hepatocellular damage in Balb C mice and pharmacological intervention by extract of Daucus carota. RADS Journal of Pharmacy and Pharmaceutical Sciences, vol. 8, no. 4, pp. 1-9.; Mughal et al., 2020bMUGHAL, T.A., ALI, S., TAHIR, H.M., MUMTAZ, S., MUMTAZ, S., HASSAN, A. and KAZMI, S.A.R., 2020b. Anti-hyperlipidemic effect of Berberis lycium Royle root bark extract in alloxanized Swiss albino mice. Punjab University Journal of Zoology, vol. 35, no. 2, pp. 261-268. http://dx.doi.org/10.17582/journal.pujz/2020.35.2.261.268.
http://dx.doi.org/10.17582/journal.pujz/...
). Mice of various groups were kept in different polypropylene cages. Ad libitum conditions were provided to the mice (adequate food and water). The temperature of the experimental room was kept within a narrow range of 22 ± 3 °C. Light and dark cycle of 12 hours was maintained in the room and the relative humidity was controlled to be within 70%.

2.9. Chemicals

Carbon tetrachloride was bought from Sigma, Aldrich (USA).

2.10. Experimental animals grouping

Twenty Swiss albino mice were obtained with an average weight of 43.6±1.5 g from Foot and Mouth Disease Research Center (FMDRC), Veterinary Research Institute (VRI), Lahore, Pakistan. The mice were treated gently, placed in cages in the hygienic and ventilated conditions in animal house. Ad libitum conditions were provided to the mice (adequate food and water). The lethal dose (LD50) of CCl4 was taken as 1 ml/kg body weight (b. w.). These animals were divided into 4 groups, each group having five mice namely I, II, III and IV. Group I was a control group administered with olive oil (0.4 ml/kg b. w.). Group II was given B. lycium Royle root bark extract (200 mg/kg b. w.). Group III was administered with CCl4 (0.4 ml/kg b. w) intra-peritoneal for a single time and group IV was given CCl4 (0.4 ml/kg b. w) and treated with B. lycium Royle root bark extract (200 mg/kg b. w.) for 14 days. All treatments were given orally. After the treatment period of 14 days, anesthesia was given to the animals after 24 hours of the last dosing, sacrificed and liver was taken for antioxidant study (Figure 2).

2.11. Estimation of antioxidants

2.11.1. Estimation of catalase

Sinha (1972)SINHA, A.K., 1972. Colorimetric assay of catalase.Analytical biochemistry, vol. 47, no. 2, pp. 389-394. method determined the catalase activity in the liver. When dichromate in acetic acid was heated with H2O2 then firstly it was converted to perchromic acid which was further converted to chromic acetate. The product was then measured at the wavelength of 620 nm. The catalase preparation was then allowed to catalyze H2O2 for various periods. Dichromate-acetic acid is added to the reaction mixture at different time intervals, stoped the reaction for sometimes and the leftover H2O2 as chromic acetate was evaluated calorimetrically.

2.11.2. Estimation of Reduced Glutathione (GSH) (non-enzymatic antioxidant)

Using the standard protocol, the activity of reduced glutathione was measured (Ellman, 1959ELLMAN, G.L., 1959. Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics, vol. 82, no. 1, pp. 70-77.). 0.5 of Elman reagent i.e., 19.8 mg DTNB dissolved in 100 mL of 0.1% sodium nitrate is added to the aliquot of 1 ml liver tissue supernatant. Following the addition of Elman reagent, phosphate buffer i.e., 3 ml was added. Then, the absorbance was calculated at 412 nm.

2.11.3. Estimation of Malondialdehyde bis-(dimethyl acetal) Tetra Ammonium (MDA)

Okkawa et al. (1979) OKKAWA, H., OHISHI, N. and YAGI, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical biochemistry, vol. 95, no. 2, pp. 351-358.described the following method. The tissues of the liver were homogenized in the solution of aqueous KC1 followed by incubation with a thiobarbituric acid reagent for about 1 hour at 90 °C. The mixture was then centrifuged after cooling. After the centrifugation, clear pink supernatant was obtained whose optical density value was determined at 532 nm. As an external standard, malondialdehyde bis-(dimethyl acetal) tetra ammonium was used.

2.11.4. Estimation of Superoxide Dismutase (SOD)

The standard method with small modifications was used to estimate the superoxide dismutase (Chia et al., 2010CHIA, R., TATTUM, M.H., JONES, S., COLLINGE, J., FISHER, E.M. and JACKSON, G.S., 2010. Superoxide dismutase 1 and tg SOD1G93A mouse spinal cord seed fibrils, suggesting a propagative cell death mechanism in amyotrophic lateral sclerosis. PloS one, vol. 5, e10627. https://doi.org/10.1371/journal.pone.0010627PMID:20498711.
https://doi.org/10.1371/journal.pone.001...
). By the addition of 1 ml of 50 mM sodium carbonate, 0.4 ml of 25 μM nitroblue tetrazolium and 0.2 ml of 0.1 mM freshly prepared hydroxylamine hydrochloride, the reaction mixture was prepared. To the reaction mixture, liver homogenate’s clear supernatant (0.1 ml, 1:10 w/v) was added. The absorbance of the sample was determined at 560 nm and changes in the value were observed.

2.12. Statistical analysis

All statistical analyses were performed by Graph Pad prism. All the values were expressed as mean ± SEM. The statistical difference among different groups was assessed by one-way ANOVA with the Bonferroni test. Values were considered statistically significant at p≤ 0.05.

3. Results

3.1. Phytochemical screening

The plant extract was tested for the presence of different phytochemicals. Results are shown in Table 2.

Table 2
Phytochemical screening.

The qualitative results are expressed as (+) for the presence and (-) for the absence of phytochemicals.

3.2. Antibacterial activity

The study was started to investigate the antibacterial effect of Berberis lycium Royle root bark aqueous extract (BLR-Ex) against various disease-causing bacteria. From the current study, it was observed that BLR extract was toxic against tested pathogenic bacteria.

Table 3 shows the zones of inhibition of BLR extract at 2.5 μg/ml, 5 μg/ml and 10 μg/mL. BLR-extract of 10μg/mL displayed antibacterial activity (E. coli: 9.3±0.7 mm; K. pneumoniae: 9.7±0.16 mm; S. aureu: 10±1.5 mm; S. pyogenes: 13.3±0.8 mm; P. seudo: 8.5±0.2 mm; S. marcesscens: 9.8±0.4 mm; S. epidermidis: 10±0.6 mm). The highest activity was shown against S. aureu (10.0±1.5 mm) and S. epidermidis (10.0±0.6 mm). 2.5 μg/ml showed minimum activity (S. aureu: 3.3±0.3 mm; S. pyogenes: 5.3±0.3 mm; P. seudo: 2±0.5 mm; S. marcesscens: 2.7±0.3 mm) and in some cases, it showed no activity at all (E. coli: 0±0 mm; K. pneumoniae: 0±0 mm; S. epidermidis: 0±0 mm) as shown in Figure 3.

Table 3
Antibacterial activity of Berberis lycium Royle extract against bacterial pathogens.
Figure 3
Zones of inhibition at 2.5, 5 and 10 µg/mL concentration of (I) DMSO and (II) BLR extract.

3.3. In vitro antioxidant activity

By the use of the DPPH test, the antioxidant capacity of extract was evaluated. Ascorbic acid was considered as the positive control. The results in Figure 4 show that antioxidant activity was increased by increasing the BLR-extract concentration. At 20, 40, 60, 80 and 100 µg/mL it was 15, 28, 35, 58, and 65% respectively (Figure 4).

Figure 4
In vitro antioxidant activity at 20-100 µg/mL.

3.4. In vivo antioxidant activity

3.4.1. Effect on Catalase (CAT)

CCl4 (0.4 mL/kg body weight) injection through intraperitoneal route caused a highly significant decrease in levels of Catalase (CCl4: 126.8±5.67 mmol/min/g liver) as compared to control (182±7.76 mmol/min/g liver). When BLR extract was given alone it caused no significant change (172.0±8.34 mmol/min/g liver) as compared to control (182±7.76 mmol/min/g liver). When BLR extract was given intraperitoneally (150 mg/kg body weight) to CCl4 treated mice, a significant change in the amount of Catalase (CCl4 + BLR extract: 163.2±3.29 mmol/min/g liver) was observed (Figure 5).

Figure 5
In vivo antioxidant activity. Analysis of catalase, GSH, MDA and SOD in the liver of Swiss albino mice. Group I: control group; Group II: B. lycium Royle root bark extract treated group; Group III: CCl4 treated group; Group IV: CCl4 plus B. lycium Royle root bark extract treated group. Keys: (b) indicates the significant difference between group I and group III. (c) indicates a significance difference between group III and group IV. Each bar signifies the mean value of five replicates and SEM. Statistical icons: c = p ≤ 0.05, bb, cc = p ≤ 0.01, bbb, ccc = p ≤ 0.001.

3.4.2. Effect on Reduced Glutathione (GSH)

CCl4 (0.4 mL/kg body weight) intraperitoneal administration resulted in a significant decrease in levels of GSH (control: 4.28±0.15 µ mol/g liver; CCl4: 2.04±0.12 µ mol/g liver). When BLR extract was given alone it caused no significant change (4.22±0.1 µ mol/g liver) as compared to control (4.28±0.15 µ mol/g liver). When BLR extract was given intraperitoneally (150 mg/kg body weight) to CCl4 treated mice, a highly significant increase in the amount of GSH (CCl4 + BLR extract: 3.68±0.42 µ mol/g liver) was observed (Figure 5).

3.4.3. Effect on Malondialdehyde bis-(dimethyl acetal) Tetra Ammonium (MDA)

Intraperitoneal administration of CCl4 (0.4 mL/kg body weight) resulted in a significant increase in levels of MDA (control: 46.2±4.45 mmol/g liver; CCl4: 652.2±20.19 mmol/g liver). When BLR extract was given alone it caused no significant change (45.8±4.95 mmol/g liver) as compared to control (46.2±4.45 mmol/g liver). When BLR extract was administered intraperitoneally (150 mg/kg body weight) to CCl4 treated mice, a highly significant decrease in the level of MDA (CCl4 + BLR extract: 349.6±31.75 mmol/g liver was observed (Figure 5).

3.4.4. Effect on Superoxide Dismutase (SOD)

Intraperitoneal administration of CCl4 (0.4 ml/kg body weight) produced a highly significant decrease in the SOD level of the CCl4 treated group (19.8±2.41 U/mg) as compared to the control (41.35±1.97 U/mg). When BLR extract alone was given to mice they caused no significant change in SOD level (40.8±1.59 U/mg). When BLR extract was injected intraperitoneally (150 mg/kg body weight) to CCl4 treated mice, the significant increase in the amount of SOD (CCl4 + BLR extract: 36.4±5.096 U/mg) was observed (Figure 5).

4. Discussion

Plant parts have been used for a long time to defeat microbes. Local populations were aware of plant’s medicinal uses. Available antibiotics face resistance to microbes. This problem is increasing day by day. Hence, the development of new antimicrobial drugs is the necessity of time. Plants are enriched with molecules that have antimicrobial activity. Plants are being scrutinized as possible sources to explore new bactericidal mediators to manage bacterial infections. Lamiaceae and Rutaceae family plants are broadly used in numerous medicinal practices to treat wide-ranging infections (Sharma et al., 2013SHARMA, U., AGNIHOTRI, R., AHMAD, S., MAHAJAN, S. and SHARMA, R., 2013. Antibacterial activity of some medicinal plants of family Lamiaceae from Braj region. Global Journal of Medicinal Plants Research, vol. 1, pp. 72-76.; Venkateshappa and Sreenath, 2013VENKATESHAPPA, S. and SREENATH, K., 2013. Potential medicinal plants of Lamiaceae. International Association of Scientific Innovation and Research, vol. 3, pp. 82-87.). Sasikala et al. (2014)SASIKALA, T., PRABAKARAN, R. and SATHIYAPRIYA, S., 2014. Evaluation of antimicrobial activities of Plectranthus barbatus L. tuber. International Journal Of Ayurvedic And Herbal Medicine, vol. 4, pp. 1506-1516. testified aqueous extract of P. barbatus against B. cereus, E. coli, S. aureus, S. epidermidis, and S. pneumonia. This extract showed antimicrobial activity against all these pathogens. Our results are in line with these results. A lot of previous studies confirmed that plants act as antibacterial agents. Results of investigations by Vatľák et al. (2014)VATĽÁK, A., KOLESÁROVÁ, A., VUKOVIČ, N., ROVNÁ, K., PETROVÁ, J., VIMMEROVÁ, V., HLEBA, L., MELLEN, M. and KAČÁNIOVÁ, M., 2014. Antimicrobial activity of medicinal plants against different strains of bacteria. Journal of Microbiology, Biotechnology and Food Sciences, vol. 3, pp. 174-176., Erecevit and Kirbağ (2017)ERECEVIT, P. and KIRBAĞ, S., 2017. Antimicrobial activity of some plant species used for the medical purpose in Turkey. The Journal of Phytopharmacology, vol. 6, no. 2, pp. 93-97. http://dx.doi.org/10.31254/phyto.2017.6205.
http://dx.doi.org/10.31254/phyto.2017.62...
, Zoysa et al. (2019)ZOYSA, M.H.N., RATHNAYAKE, H., HEWAWASAM, R.P. and WIJAYARATNE, W.M.D.G.B., 2019. Determination of in vitro antimicrobial activity of five Sri Lankan medicinal plants against selected human pathogenic bacteria. International Journal of Microbiology, vol. 2019, p. 7431439. http://dx.doi.org/10.1155/2019/7431439. PMid:31198423.
http://dx.doi.org/10.1155/2019/7431439...
, Manandhar et al. (2019)MANANDHAR, S., LUITEL, S. and DAHAL, R.K., 2019. In vitro antimicrobial activity of some medicinal plants against human pathogenic bacteria. Journal of Tropical Medicine, vol. 2019, p. 1895340. http://dx.doi.org/10.1155/2019/1895340. PMid:31065287.
http://dx.doi.org/10.1155/2019/1895340...
, Nguta and Kiraithe (2019)NGUTA, J.M. and KIRAITHE, M.N., 2019. In vitro antimicrobial activity of aqueous extracts of Ocimum suave Willd., Plectranthus barbatus andrews and Zanthoxylum chalybeum Engl. against selected pathogenic bacteria. Biomedical and Biotechnology Research Journal, vol. 3, no. 1, pp. 30-34. http://dx.doi.org/10.4103/bbrj.bbrj_128_18.
http://dx.doi.org/10.4103/bbrj.bbrj_128_...
and Othman et al. (2019)OTHMAN, L., SLEIMAN, A. and ABDEL-MASSIH, R.M., 2019. Antimicrobial activity of polyphenols and alkaloids in Middle Eastern plants. Frontiers in Microbiology, vol. 10, p. 911. http://dx.doi.org/10.3389/fmicb.2019.00911. PMid:31156565.
http://dx.doi.org/10.3389/fmicb.2019.009...
also support our findings.

It is already known that Berberis lycium bark contains an alkaloid namely Berberine which has great antimicrobial potential. The rise in the concentration of root bark extract increases the inhibitory activity (Figure 3). The poposed antibacterial mechanism of plant extract is diagrammatically expressed below (Figure 6).

Figure 6
Proposed bactericidal mechanism of plant extract.

In the current study, experiments were also designed to unveil the antioxidant potential of aqueous extract of Berberis lycium Royle’s root bark. Antioxidant activity was measured in vitro and in vivo conditions. In vitro activity was measured by the DPPH method. Antioxidant capacity relates to the reducing capability of a component. The anti-oxidant compounds are based on the reduction of the free radicals. DPPH is a stable free radical, is often involved in the determination of radical scavenging activity in the chemical analysis (Duh et al., 1999DUH, P.D., TU, Y.Y. and YEN, G.C., 1999. Antioxidant activity of water extract of Harng Jyur (Chrysanthemum morifolium Ramat). Lebensmittel-Wissenschaft + Technologie, vol. 32, no. 5, pp. 269-277. http://dx.doi.org/10.1006/fstl.1999.0548.
http://dx.doi.org/10.1006/fstl.1999.0548...
). DPPH radicals possess a single electron and at 517 nm demonstrates the highest absorption. The DPPH results are shown in Figure 4. Color removal of the DPPH solution indicates its reduced state as a result of the interaction of electrons with the reducing agents. After receiving electrons or protons DPPH free radicals were reduced (Muniyappan and Nagarajan, 2014MUNIYAPPAN, N. and NAGARAJAN, N.S., 2014. Green synthesis of silver nanoparticles with Dalbergia. Process Biochemistry, vol. 49, no. 6, pp. 1054-1061. http://dx.doi.org/10.1016/j.procbio.2014.03.015.
http://dx.doi.org/10.1016/j.procbio.2014...
). When the concentration of BLR extracts raised from 20 µg/mL to 100 µg/mL, the antioxidant activity increased up to 65% from 15%. The maximum antioxidant activity was observed as 65% at 100 µg/mL.

In vivo antioxidant activity of BLR extract was evaluated by comparing hepatic CAT, GSH, MDA and SOD levels of CCl4 intoxicated mice and BLR treated mice. In present research in CCl4 intoxicated mice level of MDA was increased while that of catalase, GSH and SOD decreased. These conditions were reversed on treatment with BLR-extract (Figure 5). These results are in line with Al-Snafi (2017)AL-SNAFI, A.E., 2017. Nutritional and therapeutic importance of Daucus carota-a review. Journal of Pharmaceutics, vol. 7, no. 2, pp. 72-88. who stated that when rats were treated with thioacetamide to induce the oxidative stress significant abate in CAT, GSH and SOD level was found while, when these mice were treated with seed extract of Dacus carota, significant elevation in the CAT, GSH and SOD level was determined according to the thioacetamide group. Rahate and Rajasekaran (2015)RAHATE, K.P. and RAJASEKARAN, A., 2015. Hepatoprotection by active fractions from Desmostachya bipinnata stapf (L.) against tamoxifen-induced hepatotoxicity. Indian Journal of Pharmacology, vol. 47, no. 3, pp. 311-315. http://dx.doi.org/10.4103/0253-7613.157130. PMid:26069370.
http://dx.doi.org/10.4103/0253-7613.1571...
had found that when female rats were intoxicated with tamoxifen to induce hepatotoxicity significant increase in the MDA level was measured according to the control group. Whereas, a polyphenolic fraction of Desmostachia bipinnata root brought down the level of MDA and elevated the total protein, SOD and GSH concentration in serum of female rats.

5. Conclusion

The present study validated that B. lycium Royle is enriched with alkaloids, steroids, tannins, saponins, glycosides, terpenoides, proteins, free amino acids, carbohydrates, phenols and flavonoids. This study also validated that B. lycium Royle root bark extract exhibits antibacterial activity. Results also revealed that hepatic damage was caused by the administration of carbon tetrachloride in Swiss albino mice. CAT, GSH, MDA and SOD levels were disturbed that were normalized by BLR extract. The present study validated that B. lycium Royle root bark extract has a potential to cope with infectious diseases and also exhibit antioxidant activity as estimated in-vivo and in-vitro.

Acknowledgments

Authors thank to the Department of Zoology, University of Azad Jammu and Kashmir, Muzaffarabad and Department of Zoology, Government College University, Lahore, Pakistan for providing all facilities for the completion of this study.

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Publication Dates

  • Publication in this collection
    22 Apr 2022
  • Date of issue
    2024

History

  • Received
    12 Mar 2021
  • Accepted
    09 Sept 2021
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