Standardization and in vitro antioxidant activity of jatamansi rhizome

Abstract

Background:

Nardostachys jatamansi Linn. commonly known as jatamansi is a well notorious drug in Indian systems of medicines having various health-related benefits and employed in various herbal formulations due to the presence of high levels of valuable phenolic constituents. The present study was aimed to quality assessment of Jatamansi rhizome by studying macro- and micro-scopic characters along with physicochemical tests, chemo-profiling using thin layer chromatography (TLC), and gas chromatography–mass spectrometry (GC-MS), in vitro antioxidant activity.

Materials and Methods:

Standardization was carried out as per the pharmacopeial guidelines and contaminant estimation was carried out by analyzing the samples for the determination of heavy metals, pesticides, and aflatoxins. Chemo-profiling was done with TLC by optimizing the mobile phase for different extracts. The GC-MS chemo-profiling was also carried out by using hexane soluble fraction of the hydroalcoholic extract. The drug is well known for a protective role in the human body as an antioxidant, so total phenolic contents and in vitro antioxidant efficacy was also determined by using established methods.

Results:

The results of quality control and anatomical studies were very much useful for its identification, whereas significant antioxidant efficacy was also observed. The drug was found free of contaminants when analyzed for pesticides and aflatoxins, whereas heavy metals were found under the pharmacopeial limit.

Conclusion:

The findings of the present research can be utilized for the identification and quality control of the jatamansi rhizome.

Nardostachys jatamansi commonly known as jatamansi is a medicinally important herb of Indian origin[1] and has been part of the traditional system of medicines to treat different disorders.[2,3,4] The various sesquiterpenes such as lignans and neolignans are present in root extracts of plant[5] and has been suggested to protect cells and tissues through its antioxidant properties.[6] It has also been reported for its protection against doxorubicin-induced lipid peroxidation.[1,7] In the present study, the quality assessment work was carried out with proper standardization and in vitro antioxidant potential.

Materials and Methods

Chemicals and plant samples

The rhizomesamples of jatamansi were purchased from local market of Delhi, India and the specimen (Ref. NISCAIR/RHMD/Consult/-2008-09/1149/181/02/01-08) authenticated by botanist Dr. H. B. Singh, Scientist F and Head Raw Material Herbarium and Museum, NISCAIR, New Delhi. All the chemicals used for the analysis and solvents were of analytical grade and obtained from Merck India. Standard catechin, ascorbic acid, and Vitamin A were purchased from Sigma Aldrich (USA).

Morphology and microscopy

The drug was evaluated for its physical appearance, shape, size, taste, color, and odor. The sample of jatamansi was cleaned and fixed in 5.0 mL formalin + 5.0 mL acetic acid + 90 mL of 70% ethyl alcohol. After 24 h of fixing, the specimens were dehydrated with graded series of tertiary butyl alcohol. Infiltrations of the specimen were carried out by gradual addition of paraffin wax (melting point 58–60°C) until t-butyl acrylate solution attained supersaturation and casted into paraffin blocks. The paraffin embedded specimens were sectioned (10–12 µm) with the help of rotary microtome and dewaxed. The sections were stained with toluidine blue, safranin and fast green, and iodine-potassium iodide.[8] Microscopic descriptions of tissues were observed with micrographs with Nikon lab photo 2 microscopic Unit (Nikon, Canada).

Physicochemical evaluation

The physicochemical studies of jatamansi rhizome were carried out according to Indian Pharmacopeia (IP, 1996).[9] The different parameters such as determination of foreign matter, loss on drying, moisture content by Karl–Fischer titration, ash values with total ash, acid insoluble ash, and water soluble ash, pH of 1% and 10% solution of jatamansi, different extractive values, were determined.

Total phenolic contents

The estimation of total phenolic contents was carried out according to the Folin–Ciocalteu (FC) method using catechin as a standard phenolic constituent.[10]

Antioxidant activity of jatamansi rhizome extract

2,2 diphenyl-1-picryhydrazyl method

The free radical scavenging efficacy of methanolic extract of jatamansi rhizome was determined using stable radical 2,2 diphenyl-1-picryhydrazyl (DPPH) method.[11] The percent inhibition of the DPPH free radical was measured using the following equation, and IC₅₀ value was calculated by using Graph prism-5.

% Inhibition = ([A₀−A₁]/A₀) × 100

where, A₀ was the absorbance of the control (blank, without extract) and A₁ was the absorbance of the extract or standard.

Nitric oxide method

Nitric oxide radical scavenging activity of jatamansi extract was determined according to the reported method.[11] The nitric oxide radicals scavenging activity was calculated as similar to DPPH method.

High-performance thin layer chromatography chemo-profiling

The high-performance thin layer chromatography (HPTLC) fingerprints of the different extracts were established by developing the solvent systems for their separation by TLC. The samples were applied 8.0 µL each with 5.0 mm width, and distance between tracks was 13 mm on precoated silica gel 60 F₂₅₄ plates (E. Merck, 0.20 mm thickness) using Linomat V, HPTLC sample applicator (CAMAG, Switzerland) and developed in the solvent system. The chromatograms were scanned at 254 and 366 nm followed by spectral analysis. Plates were also scanned in visual range after spraying visualizing reagent.

Gas chromatography–mass spectrometry profiling

Gas chromatography–mass spectrometry (GC-MS) analysis of hexane soluble fraction of the aqueous alcoholic extract was also carried out to observe nonpolar constituents.

Evaluation of contaminants

Heavy or toxic metal analysis

Different heavy metals (lead, cadmium, mercury, and arsenic) were analyzed in dried jatamansi powder by using atomic absorption spectrometer. The powdered drug (1.0 g) was incinerated in a silica crucible at a temperature 600°C until ash formation. A standard linear calibration curve was prepared with absorbance against the concentration of respective metals, and concentration was calculated in the samples.

Aflatoxins determination

The AOAC official high-performance layer chromatography HPLC method of analysis was followed for the determination of aflatoxins[12] by derivatization of dried extract and known concentration (20 ppb, 40 ppb, and 80 ppb) of standard aflatoxin B1, G1, B2, and G2. The analysis was carried out on a waters HPLC alliance e2695 separating module (Waters, USA).

Pesticide determination

The pesticides were also analyzed by using standard AOAC official GC-MS method.[12] The samples were extracted as per the method and analyzed using an Agilent 7890A GC system, USA.

Results and Discussion

Morphology and microscopy

The jatamansi rhizome was a drupe, indehiscent measuring 2.0–4.0 cm in diameter, fleshy in nature, globose in shape with longitudinal shallow furrows, consisting of yellowish green surface, whereas fleshy portion being white and spongy. The rhizome possessed strong characteristic aromatic odor with sour and astringent taste. The sample consisted of shrunken rhizome with closely appressed lateral roots. The roots and rhizome possessed only hard tissues and soft parenchymatous tissues were disintegrated. The detail microscopic characters were well depicted in Figure 1a and b.

Figure 1.

(a) TS of rhizome and large lateral roots around the rhizome, (b) TS of rhizome and enlarged portions of lateral larger root (AC- air-chambers; LS- leaf sheath; LR- lateral roots; OC- outer cortex; Scl- sclerotic pith; VC- vascular cylinder; Co-cortex; Rl-rootlets; X-xylem)

Physicochemical evaluation

Different physicochemical parameters were carried out in triplicate and are represented with standard deviation [Table 1]. The obtained results were found under limits and comparable with pharmacopeial standards.[9]

Table 1.

Summary of physicochemical parameters of jatamansi (n=3)

Total phenolic contents

Antioxidant efficacy of the plants is mainly due to the presence of phenolic constituents, which may contribute directly to the antioxidant properties. The total phenolic content in the jatamansi was measured by FC method and found to be 4.12% w/w.

Antioxidant activity of jatamansi extract

2,2 diphenyl-1-picryhydrazyl free radical scavenging activity

The jatamansi extract showed a concentration-dependent (5.0–100 μg/mL) antioxidant activity by inhibiting DPPH radical with an IC₅₀ value of 60.03 μg/mL, whereas IC₅₀ value of ascorbic acid was found to be 14.44 μg/mL, used as standard [Figure 2a].

Figure 2.

Log dose response curve of DPPH (a) and Nitric oxide (b) free radical scavenging activity of jatamansi extract compared with ascorbic acid

Nitric oxide free radical scavenging activity

The results of nitric oxide scavenging activity showed that jatamansi extract has dose-dependent moderate activity between 10 and 200 μg/mL with 81.99 μg/mL as IC₅₀ value [Figure 2b], whereas IC₅₀ value of ascorbic acid was found to be 31.36 μg/mL.

High-performance thin layer chromatography chemo-profiling

Satisfactory separation of constituents was obtained in solvent systems toluene: Ethyl acetate (73:7, v/v), petroleum ether: Diethyl ether (1:1, v/v), and benzene: Ethyl acetate (95:5, v/v) for petroleum ether, chloroform, and methanol extracts, respectively. The samples were applied, and chromatograms were developed in respective solvent systems. The chloroform extracts gave good results after sprayed with anisaldehyde sulfuric acid as visualizing agent and scanned at 530 nm [Figure 3a]. The chromatograms of petroleum ether and methanolic extracts were scanned at wavelength 254 nm [Figure 3b and c]. The HPTLC fingerprints of petroleum ether, chloroform, and methanol extract showed the presence of nine, eight, and ten constituents, respectively.

Figure 3.

Developed TLC plates and chromatograms of petroleum ether, (a) chloroform (b) and methanol (c) extracts

Gas chromatography–mass spectrometry profiling

GC-MS estimation of hexane soluble fraction of aqueous alcoholic extract showed jatamansone as major constituent [Figure 4].

Figure 4.

GC-MS chromatogram of hexane soluble fraction of jatamansi extract

Evaluation of contaminants

The quality criteria for herbal drugs are based on a clear scientific definition of the raw material. The drug should be standardized by studying physical constants and adulterations have to be checked for their identity and purity. The heavy metals, pesticide residues, and aflatoxins analysis are the very important parameters to study.

Test for heavy or toxic metal analysis

Some of the most popular Ayurvedic herbal medicinal products were found contaminated by lead, mercury, and arsenic. There are reports available on the presence of heavy metals in herbal formulations above regulatory standards considering these reports; lead, cadmium, arsenic, and mercury were analyzed in jatamansi powder and found within the limits.[9]

Aflatoxins

Aflatoxins were analyzed by using HPLC method in different samples of jatamansi. The chromatogram of mixed standard aflatoxins and sample showed that no aflatoxins are present in jatamansi sample.

Pesticides

GC is a suitable technique for such purposes, therefore; pesticide estimation was carried out by GC-MS and after comparison of chromatogram and retention time of standard thirty-one pesticides with sample, it was observed, that the test samples do not contain any pesticides.

Conclusions

The present research is guiding tool for the assessment of the quality of jatamansi rhizome on the basis of macroscopic and microscopic identification, physicochemical evaluation, TLC, and GC-MS profiling. The antioxidant activity may also be criteria to check its protecting efficacy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.