Phytochemicals, Antimicrobial and Antioxidant Potential of Methanolic Extract of Berberis aristata roots

Monika Thakur, Kunjan Sharma, Sonia Mehta, Swati Rai, Isha Sharma, Astha Tripathi*

Assistant Professor, Faculty of Applied Sciences and Biotechnology, Shoolini University, Bajhol, Solan, H.P.

*Corresponding Author E-mail: asthatripathi4u@gmail.com

ABSTRACT:

The present study focuses on exploring the presence of phytochemicals, antioxidants and antimicrobial potential of methanolic extract of root of Berberis aristata. The preliminary qualitative analysis of phytochemicals, free radical scavenging activity, antimicrobial potential and FTIR spectrophotometry were investigated from the root extracts of Berberis aristrata. Methanolic extract of root showed the presence of phytochemicals like alkaloids, carbohydrates, flavonoids, phenolic compounds and saponins. Further antioxidant assay like DPPH (Diphenyl -2-Picrylhydrazyl) assay was done. DPPH free radical scavenging activity was expressed in % inhibition. In present study, the % inhibition of methanolic extract was 52% and IC₅₀value was 26µg/ml. Antimicrobial screening showed good positive results with tested pathogens Staphylococcus aureus, Pseudomonas areginousa, Klebsiella pneumoniae and Candida albicans. The FTIR (Fourier Transform Infrared Spectrophotometer) spectroscopic studies revealed different characteristic peak values which confirmed that there is presence of various phytocompounds such as lipids, proteins in the methanolic root extracts. The FTIR method was performed on a spectrophotometer system, which was used to detect the representative peak values along with their functional groups. The results of the present study generated the FTIR spectrum profile for the medicinally important plant Berberis aristata. This preliminary study shows that root part of Berberis aristata is a promising herb with many phytochemicals, antioxidants and antimicrobial potential.

KEYWORDS: Berberis aristata, phytochemical, antimicrobial, antioxidants, FTIR, DPPH.

INTRODUCTION:

Berberis aristata is plant which is used in the herbal medicines from ancient time. Due to its therapeutic values “Rashut” is prepared by the plant for the treatment of inflammation, wound healing, skin diseases, cough, jaundice and infection of eyes^[1]. Its root, stem and leaves are extensively used in Ayurveda for the treatments of various ailments. The root of Berberis aristata contains significant amount of alkaloids chiefly berberine which having anti-inflammatory and anticancerous activities^[2]. Berberis aristata is commonly known as “Daruhaldi” and “Chitra”. This herbal plant is native to northern Himalaya’s region. In Himachal Pradesh it is commonly known as “Kashmal”^[3].

It contains various active compounds such as reducing sugars, alkaloids, flavonoids, glycosides and saponins which contributes towards its medicinal value. Alkaloids, one of the phytochemicals that have amazing effects on animal system, are a good source of antibacterial agent and powerful analgesic^[4,5]. The antimicrobial effect of the plant extracts of B. aristata may be due the presence of alkaloids such as berberin^{[6,7, 8]}. Various studies has been conducted in order to analyze its antioxidant potential. Methanolic extract of stem showed significant antioxidant potential^[9].Extracts of Berberis aristata possess substantial amount of phenols and flavonaoids with strong free radical scavenging activity^[10]. This plant is useful in treatment of various human diseases. Methanolic extract of stem showed good antimicrobial activity against various human pathogens^[11,12,13]. The selection of Berberis aristata was based upon its medicinal uses. Therefore the aim of this study was to analyze antimicrobial, antioxidant and phytochemical properties of root part of Berberis aristata.

MATERIALS AND METHODS:

Collection of plant material:

Roots of Berberis aristata plants were collected from Rohnat Village of district Sirmour, Himachal Pradesh, India, in March, 2018.

Sample Preparation:

The plant’s roots were cleaned off with tap water and unwanted materials removed by washing with running tap water and sterile water for 2-3 times. Plant roots were carefully separated and spread on sterilized blotting paper. These were then dried in hot air oven at 50-60°C for one week. The dried Berberis aristata roots were ground into fine powder.

Crude Extraction:

100grams of dried powder of B. aristata roots was extracted with methanol. Dried powder was mixed well with solvent and shaken to mix in the Sample was dipped well in the solvent, shaken and macerated in the container for 24 hours at room temperature. The lid of containers were closed tightly to prevent evaporation of methanol. After 24 hours, the solvent was collected by coarse cloth filtration and the solvent was evaporated on a water bath at 40-60°C. The concentrated crude methanolic extract was used for detailed study^[14].

Phytochemical Analysis:

Qualitative phytochemical analysis of the crude methanolic extract of B.aristata root was done by following the standard protocols^[15].

· Test for Alkaloids:

Mayer’s test was done for Alkaloids. 0.2g of extract was gently heated with 2% sulphuric acid (H₂SO₄) in a test tube for about two minutes. Four drops of Dragendorf reagent was added in extract and yielded an orange red precipitate that confirmed the presence of alkaloids.

· Test for Saponins (Frothing test):

0.2g of the extract was mixed with 1ml of distilled water. The mixture was heated to boil. Formation of a small frothy mass of bubbles implied the presence of saponins.

· Test for Flavonoids:

2g of the extract was mixed with 10ml of dimethyl sulfoxide (DMSO) and metal (lead) in a test tube. 6 drops of concentrated hydrochloric acid were then carefully added. The mixture was heated gently and formation of red color signified the presence of flavonoids.

· Test for Reducing Sugars (Fehling’s test):

Few drops of Fehling’s solution A and B were added to 10ml of extract and brick red precipitate indicated the presence of reducing sugars.

· Lead acetate test:

5ml of extract mixed with 4ml of 10% lead acetate. Bulky white precipitate was formed confirming the existence of phenolic compounds.

Antimicrobial screening:

Microbial strains Candida albicans, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus were used to check the antimicrobial activity. The bacterial cultures were sub-cultured on nutrient agar (NA) and fungal culture was grown in potato dextrose agar (PDA). Actively grown cultures were used for experiment and these cultures were preserved at 4°C for further use.

Antimicrobial activity using Agar well diffusion method:

Antibacterial and antifungal activities of the methanolic extracts B. aristata root were tested using agar well diffusion method. 100mg/ml extract of B. aristata root was dissolved in 1ml of dimethyl sulfoxide (DMSO). Actively grown bacterial and fungal strains were then carefully swabbed over the respective media plates using sterilized cotton swabs for a uniform distribution of the microbial cultures. 5mm diameters wells were made using sterile cork-borer in these inoculated plates and wells were filled with 80µl of methanolic extract. DMSO and Ciprofloxacin and Fluconazole were used as positive control for bacteria and fungi, respectively. DMSO was used as negative control. The plates with bacterial cultures were incubated overnight at 37°C and those for fungi at 28°C for 72 hours. The antimicrobial activities of methanolic extract were evaluated by measuring diameter of zone in millimeters (mm). The experiment was performed in triplicates and means were calculated ^[8].

Antioxidant activity:

DPPH radical-scavenging activity:

Antioxidant activity was evaluated by DPPH radical scavenging method. Free radical scavenging activity of methanolic extract was measured by 1, 1- diphenyl-2-picryl hydrazyl (DPPH). In brief, 0.1mM solution of DPPH in methanol was prepared. 1ml solution of DPPH was mixed with 3ml B. aristata roots extract at different concentration (10, 20, 30, 40µg/ml). The mixture was shaken well and allowed to stand at room temp for 30 minutes. Absorbance was measured at 517nm by using spectrophotometer. The IC₅₀ value of the sample, which is the concentration of sample required to inhibit 50% of the DPPH free radical, was calculated using Log dose inhibition curve. Lower absorbance of the reaction mixture indicated higher free radical activity. DPPH scavenging effect was calculated by using following equation: DPPH scavenging effect (%) or Percent inhibition = A_O – A₁ / A_O × 100. Where A_O was the Absorbance of control reaction and A₁ was the Absorbance in presence of test sample.

FTIR (Fourier Transform Infrared Spectrophotometer) analysis:

Dried powder of methanolic extract of B. aristata root was used for FTIR analysis. 10mg of the dried extract powder was encapsulated in 100mg of KBr (Potassium Bromide) pellet, in order to prepare translucent sample discs. The powdered sample of methanolic extract of plant root was loaded in FTIR spectroscope with a Scan range from 400 to 4000cm^-1 with a resolution of cm^-1 at room temperature.

Statistical Analysis:

All the experimental analysis was carried out in triplicates. The results are expressed as mean values and standard deviation (SD). The results were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD Test using SAV v.9.1.3 program. Differences at p< 0.05 were considered to be significant.

RESULTS:

Root part of Berberis aristata is crushed into fine powder and stored in an airtight container for further experimental work.

Phytochemical screening:

In current study, qualitative analysis of phytochemicals has been done and preliminary screening showed occurrence of alkaloids, carbohydrates, flavonoids, phenolic and saponins depicted in Fig.1 and Table-1.

Table 1: Qualitative analysis of Phytochemical present in methanolic extract of Berberis aristata root

S. No	Phytochemicals	Chemical tests	*Berberis aristata*
1.	Alkaloids	Mayer’s test	+++
2.	Carbohydrates	Benedict’s test	+
3.	Flavonoids	Alkaline reagent Test	++
4.	Phenolic compounds	Lead acetate test	+++
5.	Saponins	Saponins test	++

*Weakly present (+), moderate (++), strongly present (+++)

Antimicrobial assay:

Antimicrobial activity of methanolic extract of B. aristata root was tested against 3 bacterial and 1 fungal strains. Ciprofloxacin and fluconazole were used as positive control for bacterial and fungal strains, respectively. DMSO (Dimethyl sulfoxide) was used as negative control. 100mg/ml extract was used and experiment was performed in triplicates (Fig.2). The methanolic extract B. aristata root showed promising antimicrobial activity against all the tested strains. Maximum zone of inhibition was recorded against Candida albicans followed by Staphylococcus aureus (Table-2).

Table 2: Zone of inhibition (mm ± SD) of methanolic extract of B. aristata root

S. No.	Microorganisms	Zone of inhibition (mm)
S. No.	Microorganisms	Methanolic extract of *B. aristata* root	Positive control
1	S. aureus	22 ±0.088^ab	20±0.4^ab
2	C. albicans	25 ±0.033^a	24±0.0.23^a
3	P. areuginosa	20 ±0.046a^ab	18±0.0.11^b
4	K. pneumonia	19 ±0.057^b	10±0.75^c

*In each column different letters means significant difference (p<0.05)

Antioxidant activity of methanolic extract of Berberis aristata root:

In present study it was found that percentage inhibition of methanolic extract of Berberis aristata root is 52%. IC₅₀ value came out to be 26µg/ml. The Percentage inhibition was increased with increase in the concentration of extract showed in Table-3 and Fig. 3.% inhibition of DPPH corresponding to the concentration of methanolic extract was observed as below. In present study, DPPH free radical scavenging assay in order to determine the radical scavenging ability of root extract. This assay is widely used widely used in vitro assays for determining the radical scavenging effect of extracts. In the presence of an extract which has capability to donate hydrogen atom, the free radical nature of DPPH is lost and the color (purple) changes to yellow (diphenylpicrylhydrazine).

Table 4: FTIR analysis of methanolic extract of Berberis aristata root

S. No.	Wave no. (cm-¹) Test sample	Wave no. (cm-¹) Reference	Function group	Phytocompounds identified
1.	3371	3570- 3200	O-H Stretch, Hydroxy group, H- Bonded	Polyhydroxy compound
2.	2926	2935- 2915	Asymmetric stretching of –CH (CH2) vibration	Lipids, proteins
3.	2855	2865- 2845	Symmetric stretching of –CH (CH2) Vibration	Lipids, proteins
4.	2263	2300- 1990	Multiple bonding	Nitrile compound
5.	1737	1740- 1725	C=O Stretch	Aldehyde compound
6.	1603	1650- 1600	C=O Stretch	Ketone compound
7.	1508	1510- 1450	C=C-C, Aromatic Ring	Aromatic compound
8.	1474	1510- 1450	C=C-C, Aromatic Ring	Aromatic compound
9.	1389	1410- 1310	O-H Bend, Alcoholic Group	Phenol or Tertiary Compound
10.	1274	1340- 1250	CN Stretch	Aromatic primary Amine
11.	1102	1140- 1070	C-O Stretch, ether Group	Cyclic ethers
12.	1033	1100- 1000	PO3 Stretch	Phosphate ion
13.	914	995- 850	P-O-C	Aromatic phosphate
14.	871	995- 850	P-O-C	Aromatic phosphate
15.	821	850- 800	Alkyl halide, aliphatic Amine	Aliphatic amine

Fig. 4: FTIR spectroscopy of methanolic extract of Berberis aristata root

DISCUSSION:

Different studies have been done by the researchers confirmed that the antimicrobial potential of B. aristata is worldwide acceptable for its traditional uses^[16]. Root, stem and fruits of B. aristata have been reported against various pathogenic bacteria^[7,17,18]. The findings of the present study revealed that the 100mg/ml methanolic extract of B. aristata root showed effective antimicrobial activity against all tested bacteria and fungi. Qualitative phytochemical screening showed positive tests for all phytochemicals like alkaloids, flavonoids, saponins, terpenoids, steroids and reducing sugars^[19]. Pharmacological studies conducted in various part of India reveals that this plant has proven activity as an antioxidant^[20,21].

CONCLUSION:

The methanolic extract of B. aristata root contains various phytochemical compounds and FTIR analysis confirmed the presence of these bioactive compounds. In comparison to antibiotics the methanolic extract of B. aristata root showed higher zone of inhibition against all tested microorganisms. Thus, this plant extract can be used as a good replacement to the harmful antibiotics. Antioxidant assay showed the free radical scavenging activity of methanol root extract of B. aristata. The results suggested that the methanolic extract of B. aristata root posses novel compounds with antimicrobial properties which can be further used for drug discovery and structural elucidation will also required which will be done by advanced spectroscopic.

CONFLICT OF INTEREST:

The authors have no conflicts of interest.

ACKNOWLEDGEMENT:

The authors acknowledge Shoolini University, Solan, for providing infrastructure support to conduct the research work. Authors also acknowledge the support provided by mycology laboratory, school of biotechnology, Shoolini University, Solan, India.

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Received on 14.12.2019 Modified on 02.03.2020

Research J. Pharm. and Tech. 2020; 13(12):5763-5767.

DOI: 10.5958/0974-360X.2020.01004.5

Sample	Concentration of methanolic extract used (µg/ml) (Scavenging activity of free DPPH radicals) (%)
Sample	10	20	30	40	50
Berberis aristata	12±0.03 ^a	35±0.02 ^b	43±0.03 ^b	52±0.48 ^b	60±0.32 ^b
Ascorbic acid	10.56±0.12 ^c	32.71±0.06 ^b	48.46±0.02 ^a	66.33±0.01 ^a	83.57±0.01 ^a