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Table of Contents
Year : 2019  |  Volume : 2  |  Issue : 3  |  Page : 141-146

Investigation of the pharmacognostical, phytochemical, and antioxidant studies of various fractions of Dichrostachys cinerea root

1 Department of Pharmacognosy, Central Ayurveda Research Institute for Drug Development, CCRAS, Ministry of AYUSH, Kolkata, West Bengal, India
2 Department of Pharmacognosy, G. Pulla Reddy College of Pharmacy, Mehdipatnam, Hyderabad, Telangana, India
3 Faculty of Pharmacy, University College of Chemical Technology, Osmania University, Hyderabad, Telangana, India

Date of Web Publication1-Jul-2019

Correspondence Address:
Rajesh Bolleddu
Department of Pharmacognosy, Central Ayurveda Research Institute for Drug Development, CCRAS, Ministry of AYUSH, Government of India, Kolkata - 700 091, West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JNSM.JNSM_56_18

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Aim: The objective of this study was to analyze the pharmacognostical, phytochemical and antioxidant studies of aqueous ethanolic extract, petroleum ether, chloroform, ethyl acetate and butanol fractions of Dichrostachys cinerea root (Veerataru). Materials and Methods: The pharmacognostical studies on D. cinerea root including parameters such as powder microscopy, extractive values, ash values, and fluorescence and the phytochemical studies are established. The aqueous ethanolic extract and all the fractions were screened against few free radicals, such as diphenylpicrylhydrazyl radical, hydroxyl radical, nitric oxide radical and superoxide anion radical. Results: Powder microscopy revealed the presence of lignified crystal fibers, stone cells, and prism-shaped crystals. All the fractions were rich in steroids, flavonoids, phenolic compounds, and carbohydrates. High level of total phenolic content (158 mg gallic acid equivalent/g) and flavonoids (32 mg rutin equivalent/g) was observed in ethyl acetate fraction. Conclusions: It is concluded that the ethyl acetate fraction followed by butanol fraction of D. cinerea root has strong antioxidant potential. Further study is required for the isolation of bioactive component, which may serve as a potent natural antioxidant.

Keywords: Antioxidants, Dichrostachys cinerea, diphenylpicrylhydrazyl, phenolic compounds, stone cells

How to cite this article:
Bolleddu R, Venkatesh S, Rao M M, Shyamsunder R. Investigation of the pharmacognostical, phytochemical, and antioxidant studies of various fractions of Dichrostachys cinerea root. J Nat Sci Med 2019;2:141-6

How to cite this URL:
Bolleddu R, Venkatesh S, Rao M M, Shyamsunder R. Investigation of the pharmacognostical, phytochemical, and antioxidant studies of various fractions of Dichrostachys cinerea root. J Nat Sci Med [serial online] 2019 [cited 2023 Jan 30];2:141-6. Available from: https://www.jnsmonline.org/text.asp?2019/2/3/141/254481

  Introduction Top

Free radicals are highly unstable chemical molecules with lone pair of electron and react with other compounds to capture the needed electrons to gain stability. Lipids' auto-oxidation and reactive nitrogen species are the main sources of free radicals in the form of hydroxyl radicals (OH·¯), nitric oxide radicals (NO·), superoxide anions (O2·¯), and hydrogen peroxide (H2O2).[1] Free radicals can also be generated by smoking, ultraviolet radiation, environmental pollutants, and medication, which are likely to damage low-density lipoproteins (LDLs), cell proteins, and DNA.[2],[3] These reactive oxygen species can cause diseases such as cancer, rheumatism, insomnia, diabetes, asthma, kidney disorders, and atherosclerosis.[4],[5] Polyphenols are natural antioxidants, acts as a free radical scavenger and prevents the cell damage, hence called as cell saviors.[6] Several medicinal plants possessing higher polyphenols which may act as natural antioxidants.[7] Synthetic antioxidants are reported to possessing carcinogenic actions, while natural antioxidants have considerably increased for use in pharmaceutical products, cosmetics, and food.[8]Dichrostachys cinerea (L.) Wight. and Arn.(Mimosaceae) is used to cure rheumatism, strangury, diabetes, urinary calculi, and renal troubles in Indian traditional medicine. It is commonly called as “veeratharu” in Ayurveda.[9] Roots are used in treatment of asthma,[10] it also possess diuretic[11] antibacterial[12] and nephroprotective activities.[13] Stem bark has antidiabetic[14] and anti-inflammatory properties.[15] Leaves possesses antimicrobial[16] and antidiarrheal activities.[17]

  Materials and Methods Top

Collection of roots and preparation of plant extract

The D. cinerea roots were collected from Anajipuram (V), Suryapet (Dist.), Telangana, India. Authentication of the plant was done by Taxonomist, Botanical Survey of India, Hyderabad. Voucher specimen of D. cinerea is GPRCP/DC/BR07/2015 maintained. The roots were shade dried and ground into powder. The dried root powder is used for the microscopical and physicochemical investigations. Root powder (2.4 kg) was macerated with ethyl alcohol (80%) for 8 days. The solvent was separated from the extract in a rotavapor and dried. The final yield of aqueous ethanolic extract (mother extract of D. cinerea root [MDCR]) is 120 g.

Powder microscopy

The D. cinerea root powder was treated with chloral hydrate reagent. To sample on microscopic slide, 1–2 drops of phloroglucinol reagent was added and a coverslip was placed above the sample. Then, microscopic slides were mounted and tracing was done.[18]

Physicochemical studies

Physicochemical parameters such as ash values, extractive values, loss on drying, and fluorescence analysis of root powder were determined according to standard methods. Air-dried sample was used for all these quantitative studies.[19]

Fractionation of extract

To the aqueous ethanolic extract, distilled water up to 500 ml was added, and whole content was transferred into separating funnel and fractionated with petroleum ether, chloroform, ethyl acetate, and n-butanol (4 × 500 ml with each solvent). The percentage yields of petroleum ether (petroleum ether fraction of D. cinerea root [PDCR]), chloroform (chloroform fraction of D. cinerea root [CDCR]), ethyl acetate (ethyl acetate fraction of D. cinerea root [EDCR]), n-butanol (butanol fraction of D. cinerea root [BDCR]), and remaining aqueous fraction (aqueous fraction of D. cinerea root [ADCR]) of MDCR were 1.8%, 4%, 12%, 30%, and 30%.[20]

Phytochemical screening

The hydroalcoholic extract and fractions of D. cinerea were tested for the detection of phenols, flavonoids, alkaloids, glycosides, carbohydrates, proteins, steroids, and saponins.[21],[22]

Free radical scavenging studies

Determination of diphenylpicrylhydrazyl radical scavenging activity

According to Aquino et al.'s method,[23] to the 2 ml of 90 μM of diphenylpicrylhydrazyl (DPPH) solution, 1 ml of different concentrations of D. cinerea extracts, reference compounds ascorbic acid and curcumin were added in individual test tubes. Incubate all the test tubes in dark condition for 1 hour. Add 1 ml of ethanol to each test tube. Absorbance was read at 517 nm. IC50 was determined by plotting graph between concentrations versus Percentage of radical scavenging capacity (%RSC).


Determination of nitric oxide radical scavenging activity

According to Griess reactions,[24] to 1 ml of different fractions of D. cinerea and curcumin, ascorbic acid-positive controls, 0.5 ml of 5 mM sodium nitroprusside was added. After incubation, 1.5 ml of incubated mixture is diluted with 1.5 ml of Griess reagent. The absorbance was measured at 540 nm. The %RSC measured by comparing the absorbance value of control and test; IC50 was determined.

Determination of superoxide anion radical scavenging activity

According to Nishikimi et al.'s method,[25] absorbance of test and reference compounds was measured at 560 nm against control. Gallic acid and rutin were used as reference compounds. The %RSC and IC50 values were determined.

Determination of hydroxyl radical scavenging activity

According to Kunchandy and Rao method, OH· generated in Fenton reaction reacts with deoxyribose and forms malondialdehyde, which further reacts with thiobarbituric acid (TBA) and forms pink chromogen.[26] The absorbance of test and standard solutions was measured at 532 nm against control containing deoxyribose and buffer. Butylated hydroxyl toluene (BHT) and mannitol were used as standards.

Determination of reducing power

The reducing power of mother extract and all the fractions were determined according to the method of Oyaizu.[27] Absorbance of all samples was measured at 700 nm. Curcumin and BHT were used as reference compounds. Higher absorbance of reaction mixture indicates greater reducing power.

Determination of total phenolic content

To 0.5 ml of test solution, 5 ml of 10% Folin–Ciocalteu (F-C) reagent and 4 ml of 1 M of aqueous sodium carbonate were added. After 15 min, the absorbance was measured at 765 nm. Standard curve of gallic acid (10–100 μg/ml) was plotted by absorbance versus concentration.[28]

Determination of total flavonoid content

Total flavonoid content is determined by aluminum chloride method.[29] The standard graph was plotted with the series of rutin concentrations (10–100 μg/ml) on X-axis and the absorbance on Y-axis. The total flavonoids are expressed as rutin equivalents per gram.

  Results and Discussion Top

Powder microscopy

Root powder is reddish brown, with no specific odor and taste. The results of powder microscopy are shown in [Figure 1]. It shows diagnostic microscopic characters such as different sizes of lignified stone cells with large lumen, thick-walled cork cells, prismatic calcium oxalate crystals, lignified crystal fibers, and thin-walled peridermal cells.
Figure 1: Powder microscopy of root: (a) cork, (b and c) crystal fibers, (d) parenchyma cells, (e) phloem fiber, (f) calcium oxalate crystals, (g) peridermal cells, (h and i) stone cells

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Physicochemical studies

The results of physicochemical parameters are shown in [Table 1]. The total ash and acid-insoluble ash values were 3.85% and 0.3%, respectively. The extractive values for various solvents such as ethanol and water were found to be 33.04% and 26.44%. The moisture content was calculated through loss on drying method and the value was found to be 9.2%.
Table 1: Physicochemical parameters

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Fluorescence analysis

In different solvents, various color radiations are emitted for root powder. These color radiations based on solvents useful as a voluble diagnostic tool for proper authentication of D. cinerea root powder. Data are represented in [Table 2].
Table 2: Fluorescence analysis of Dichrostachys cinerea root powder

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Preliminary phytochemical screening

[Table 3] represents the results of phytochemical screening of D. cinerea extracts. The results indicate the presence of steroids, flavonoids, polyphenols, and carbohydrates.
Table 3: Phytochemical analysis of various fractions of Dichrostachys cinerea

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Diphenylpicrylhydrazyl radical scavenging activity

At 517 nm, DPPH in its radical form has an absorption peak which disappears by an antioxidant due to reduction mechanism. IC50 values indicated that different extracts of D. cinerea showed good DPPH radical scavenging activity, which is shown in [Table 4]. Ethyl acetate fraction of D. cinerea root (IC50 of EDCR-6.7 μg/ml), mother extract of D. cinerea root (IC50 of MDCR-8 μg/ml) DPPH radical scavenging capacity is comparable to standard curcumin (IC50-5.5 μg/ml). The decreasing order of DPPH radical scavenging activity is ascorbic acid > curcumin > EDCR > MDCR > BDCR > ADCR > CDCR > PDCR.
Table 4: IC50 values of various test and standard drugs (μg/ml)

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Nitric oxide radical scavenging activity

Nitric oxide is generated from sodium nitroprusside and rapidly oxidized to nitrite and nitrate ions. Under acidic conditions, this nitrite ion oxidizes sulfanilamide to form diazonium, which coupled with naphthyl ethyl diamine dihydrochloride to form pink dye which was measured at 540 nm. Standard curcumin and EDCR showed good nitric oxide radical scavenging activity in concentration range of 10–100 μg/ml and decreasing order of IC50 values was EDCR > curcumin. Standard ascorbic acid, MDCR, ADCR, and BDCR showed good nitric oxide radical scavenging activity in concentration range of 100–1000 μg/ml and decreasing order of IC50 values was ascorbic acid > BDCR > MDCR > ADCR.

Super oxide radical scavenging activity

Molecular oxygen produces superoxide radicals due to oxidative enzymes of the body and auto-oxidation reactions by catecholamines. These generated superoxide radicals reduce NBT to blue color which was measured at 560 nm.[30] Standard gallic acid inhibited the superoxide radicals in concentration range of 10–100 μg/ml. All extracts of D. cinerea exhibited a superior activity than standard rutin. However, the activity is not comparable with gallic acid (IC50= 35 μg/ml). The decreasing order of superoxide radical scavenging activity was gallic acid > EDCR > CDCR > BDCR > MDCR > rutin, ADCR.

Hydroxyl radical scavenging activity

Hydroxyl radicals can cause serious oxidative damage to proteins, lipids, and DNA. Hydroxyl radicals are detected by degradation of 2-deoxy-2-ribose into fragments which on heating with TBA forms pink chromogen.[31] All the fractions do not show any promising hydroxyl radical scavenging property even at the concentrations up to 1000 μg/ml, whereas standard mannitol and BHT have shown good hydroxyl radical scavenging activity, with IC50 values 43 and 145 μg/ml.

Assay of reducing power

The results are shown in [Figure 2]. Increase of absorbance values indicates increased reducing power activity. The reaction involves conversion of Fe+3 to Fe+2 in the presence of antioxidant. Standard curcumin, BHT, and test drug MDCR have shown good reductive capabilities in concentration range of 1–10 μg/ml. EDCR has shown good reductive capabilities in concentration range of 10–100 μg/ml. ADCR, BDCR, CDCR, and PDCR have shown good reductive capabilities in concentration range of 100–1000 μg/ml. The decreasing order of reductive capabilities was BHT > MDCR > curcumin > EDCR > BDCR > ADCR > CDCR > PDCR.
Figure 2: Reducing power of different fractions of Dichrostachys cinerea and standard drugs. BHT: Butylated hydroxyl toluene, MDCR: Mother extract of Dichrostachys cinerea root, PDCR: Petroleum ether fraction of Dichrostachys cinerea root, CDCR: Chloroform fraction of Dichrostachys cinerea root, EDCR: Ethyl acetate fraction of Dichrostachys cinerea root, BDCR: Butanol fraction of Dichrostachys cinerea root, ADCR: Aqueous fraction of Dichrostachys cinerea root

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Estimation of total phenolic content

Total phenolic content was determined by F-C assay using gallic acid as a standard phenolic compound. Among all fractions, ethyl acetate fraction showed highest phenolic content followed by butanol fraction. The total phenolic content is shown in [Table 5].
Table 5: Phenolic and flavonoid content of all fractions

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Estimation of total flavonoid content

Total flavonoid content of mother extract and all the fractions was determined by aluminum chloride assay using rutin as a standard flavonoid compound. The total flavonoid content was found to be higher in EDCR and BDCR among all fractions.

  Conclusions Top

The present investigation established the qualitative and quantitative diagnostic features of roots of D. cinerea through powder microscopical, physicochemical, phytochemical, and fluorescence analyses. Phytochemical analysis revealed that all the fractions are rich sources of saponins, phenols, flavonoids, carbohydrates, and proteins. Higher level of antioxidant activity is observed in ethyl acetate fraction, followed by butanol fraction and ethanol extract when compared to other extracts. The nitric oxide radical scavenging capacity of ethyl acetate fraction is greater than standard curcumin and ascorbic acid. It has shown considerable superoxide and DPPH radical scavenging capacity and IC50 values are near to standard values. Among the entire extracts, ethyl acetate fraction is found to be rich in total phenolic and flavonoid content. These constituents may be responsible for observed antioxidant potential. The ethyl acetate extract can be considered a new source of natural antioxidant.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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