Int J Pharm Pharm Sci, Vol 9, Issue 10, 205-210Original Article


SCREENING OF TOTAL PHENOL AND FLAVONOID CONTENT IN DIFFERENT CYTOTYPES OF TWO SPECIES OF ACHYRANTHESLINN. FROM WESTERN HIMALAYA,INDIA

MANJIT INDER SINGH SAGGOO1, LOVLEEN2

1Department of Botany, Punjabi University, Patiala 147002, Punjab, India, 2Department of Botany, DAV College, Jalandhar 144008, Punjab, India
Email: ms.lovleen7@gmail.com

Received: 07 May 2017 Revised and Accepted: 31 Aug 2017

ABSTRACT

Objective: Genus AchyranthesLinn. belonging to family Amaranthaceae consists of six species. The present study was undertaken to screen the phenolic components in the different cytotypes of two species of Genus AchyranthesLinn. growing in western Himalaya, India.

Methods: Methanol extract of leaves was used to determine the total phenol and flavonoid contents in different cytotypes of A. aspera Linn. and A. bidentataBlume by spectrophotometric method. Total phenol content was expressed as mg gallic acid g-1phenol and total flavonoid content as mg quercetin g-1flavonoid using the standard curves. Further, gallic acid content in methanol extracts of leaves was determined by RP-HPLC method using C-18 column, employing 0.01% (v/v) orthophosphoric acid: acetonitrile (98:2 v/v) as mobile phase at a flow rate of 1 ml/min with ultraviolet (UV) detection at 272nm.

Results: Hexaploid plants of A. aspera Linn. possess the higher amount of phenol (9.16±0.84 mg/g) and flavonoid (78.36±1.63mg/g) constituents in the methanol extract of leaves as compared to its dodecaploid counterparts (7.86±0.08mg/g and 70.20±1.81mg/g respectively). Similarly, phenol and flavonoid contentis found to be more in the methanol extract of leaves of hexaploid plants of A. bidentataBlume (11.93±0.59mg/g and 115.92±1.32mg/g respectively) as compared to its dodecaploid counterparts (9.46±0.75mg/g and 107.76±0.94mg/grespectively). Further, RP-HPLC analysis of gallic acid revealed that higher amount of gallic acid is present in dodecaploid plants of A. aspera Linn. (1.04±0.02 mg/g) and A. bidentataBlume (1.34±0.03 mg/g) as compared to hexaploid counterparts (1.01±0.01mg/g and 1.22±0.05mg/g respectively).

Conclusion: The present investigation revealed that A. aspera Linn. and A. bidentataBlumeplants show immense intraspecific variability in their phenolic components. Hence there is need to evaluate germplasm to select superior genotype for medicinal and conservation purpose.

Keywords: AchyranthesLinn, Cytotypes, Total phenols,Total flavonoids, Western Himalaya

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2017v9i10.19780

INTRODUCTION

Achyranthes is a genus of about six species in tropical and subtropical regions, mostly in Africa and Asia [1]. Out of three species of the genus available in India, A.asperaLinn. is distributed throughout India and commonly found on hillsides, waste places, roadsides and riverbanks up to the ranges of 2300m while A. bidentataBlume grows in temperate and subtropical Himalaya from Kishtwar to Sikkim, Khasia Hills, Bihar, Konkan, Nilgiris and Travancore at an elevations of 1200 m-2400m.

Traditionally, A. asperaLinn.is used in asthma, cough, oedema, dropsy, piles, boils, eruptions of skin, pneumonia, snake bites and night blindness. It is pungent, antiphlegmatic, antiperiodic, diuretic, purgative and laxative [2]. Ash of the plant is applied externally to ulcers and warts. The crushed leaves rubbed on aching back cure strained back [3]. A fresh piece of root is used as tooth brush. A paste of the roots in water is used in ophthalmia and opacities of the cornea. A paste of fresh leaves is used for allaying pain from thebite of wasps [4]. Inhaling the fumes of A. asperamixed with Smilax ovalifoliaroots is suggested to improve appetite and to cure various types of gastric disorders [5]. The plant is useful in liver complaints, rheumatism, scabies and other skin diseases.

As an important medicinal herb, the plant of A. bidentataBlume is used againstwhooping cough, abdominal pain,backache, trauma, pain and difficulty in movement of knees, blisters in the mouth and cholera, scorpion sting and malarial fever. The plant is diuretic and astringent [6-8].

Further, aperusal of the literature revealed the occurrence of different cytotypes among these species. Therefore, the present investigation has been taken up to evaluate these species with immense medicinal importance for their bioactive contents depending upon their different ploidy levels. The present study is the first attempt to carry out the investigation of total phenol,total flavonoid and gallic acid content in different cytotypes of A. aspera Linn. and A. bidentataBlume.

MATERIALS AND METHODS

Collection of plant material

The healthy plants of A. aspera Linn. and A. bidentataBlume were collected from different localities in western Himalaya of India (table 1). Identification of the specimens was done at Botanical Survey of India, Dehra Dun, India. Voucher specimens have been deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN).

Table1: Chromosome number, ploidy,locality with altitude and accession number (PUN) of different cytotypes of Achyranthesaspera Linn and AchyranthesbidentataBlume from western Himalaya, India

Taxa Chromosome number(2n) Ploidy Locality with altitude (m) Accession number (PUN)
A.aspera Linn. 42 Hexaploid Tathal (Himachal Pradesh), 1100m 59410
A.aspera Linn. 84 Dodecaploid Dheera (Himachal Pradesh), 500m 59411
A.bidentataBlume 42 Hexaploid Dharamkot (Himachal Pradesh), 2100m 58572
A.bidentataBlume 84 Dodecaploid Rehlu (Himachal Pradesh), 950m 58575

Chemicals and reagents

Acetic acid (Fisher Scientific), chloroform (Merck), ethanol (Changshu Yangyuan Chem), acetocarmine (BDH), Folin-Ciocalteu reagent (Loba Chemie), anhydrous Na2CO3 (S D Fine Chem),NaNO2 (Sarabhai M Chemicals), AlCl3 (S D Fine Chem) and NaOH (Loba Chemie) used were of analytic grade. Methanol (Merck), gallic acid (HiMedia), quercetin (Sigma-Aldrich),orthophosphoric acid (Loba Chemie) and acetonitrile (Merck) used were of HPLC grade.

Meiotic analysis

For meiotic analysis, the young floral buds of suitable size were fixed in Carnoy’s fixative (6 alcohol: 3 chloroform: 1 acetic acid v/v/v) for 24 h and then preserved in 70% alcohol at 4˚C until use. For the cytological studies, anthers were squashed in 2% acetocarmine. A number of slides were carefully examined for chromosome counts. Photomicrographs of chromosome counts were made from freshly prepared slides using 80i Eclipse Microscope.

Extract preparation

The leaves of the selected plants were washed thoroughly and air dried in shade at room temperature. The dried leaves were ground into acoarse powder with a mechanical grinder and stored inair tight containers at room temperature for further use. 1g powder of each cytotype was suspended in 50 ml methanol for 48h at room temperature with occasional stirring. After 48h, methanol extract was filtered with Whatman no. 1 filter paper.The filtrate was saved and allowed to dry at 37˚C in an incubator for complete evaporation of methanol. The whole process was repeated three times and finally blackish-green coloured, concentrated leaf extract was obtained. Dried extract was weighed and dissolved in methanol to make afinal concentration of 100 mg/ml of extract and stored in therefrigeratoruntil further use.

Determination of total phenols

Total phenol contentdetermination was performed by using a modified Folin-Ciocalteu reagent method [9]. Folin-Ciocalteu reagent was diluted to 10 fold with double distilled water. 0.1ml of methanol extract of leaves was mixed with 7.9ml of double distilled water. Then, 0.5 ml of diluted Folin Ciocalteu reagent was added and incubated for 3 min at room temperature. After that, 1.5 ml of 20% of anhydrous Na2CO3 (w/v) was added and mixed thoroughly. The mixture was incubated at room temperature for 2 h. The absorbance of the sample was recorded at 765 nm using spectrophotometer (Shimadzu, UVmini-1240 UV-VIS Spectrophotometer) against ablank containing double distilled water instead of leaf extract.The experiment was conducted in triplicate. The total phenol content was determined as mg gallic acid g-1phenol using the standard curve.

Determination of total flavonoids

Total flavonoid content was determined by themethod proposed by Kim et al. [10]with minor modifications. 1ml of methanol extract of leaves was mixed with 4ml of double distilled water. Then, 0.3ml of 5% NaNO2 (w/v) was added and incubated for 5 min at room temperature. Later, 0.3ml of 10% AlCl3 (w/v), 2ml of 1M NaOH and 2.4ml of double distilled water was added. The mixture was incubated up to 30 min at room temperature and absorbance was measured at 510 nm using spectrophotometer (Shimadzu, UVmini-1240 UV-VIS Spectrophotometer). The experiment was performed in triplicate. The total flavonoid content was determined as mg quercetin g-1flavonoid using the standard curve.

RP-HPLC conditions

Reverse phase-high performance liquid chromatography (RP-HPLC) was used for the detection of gallic acid.RP-HPLC was performed by modifying the method proposed byRakeshet al.[11].HPLC (Shimadzu) C-18 column with dimensions 250 mm x 4.6mm and particle size 5μmwas used. The column temperature was maintained at 30 ˚C. Mobile phase used was 0.01% (v/v) orthophosphoric acid: acetonitrile (98:2 v/v) with a flow rate of 1 ml/min. The injection volume was 20 μl and detecting wavelength was 272nm. The mobile phase was filtered through a0.45μm membrane filter.

RESULTS AND DISCUSSION

Chromosome number

The meiotic analysis was carried out on different populations of A. aspera and A. bidentata from western Himalaya, India.The existence of two different chromosome counts of 2n=42, 84 (fig.1) was observed in both the studied species of Achyranthes.The species are hexaploid (2n=42) and dodecaploid (2n=84) based on base number x=7[12]. The present chromosome counts are in conformity with the earlier reports from India and elsewhere [13-38]. However, varied chromosome reports have also been reported earlier for A. asperai.e. 2n=14, 36, 48 and 96[39-41]and A. bidentatai.e. 2n=34, 35, 36, 38 [36, 42].

Fig.1:A-B: Achyranthes asperaLinn.:A. A PMC at metaphase-I showing 21 bivalents,B. A PMC at metaphase-I showing 42 bivalents; C-D: Achyranthes bidentata Blume: A PMC at diakinesis stage showing 21 bivalents,b. A PMC at metaphase-II showing 42:42 distribution of chromosomes. Scale bar=10μm

Phenol and flavonoid estimation

The amount of phenol and flavonoid contents in leaf extracts of A. aspera and A. bidentatawereestimated by spectrophotometric method. Gallic acid was used as a standard compound and total phenoliccontent was expressed as mg gallic acid g−1 usingthe equation based on thecalibration curve (fig. 2): y=0.0007x, R2=0.9946, where y is an absorbanceand x is mg gallic acid g−1. The total flavonoid contentwas expressed as mg quercetin g−1 using the straight lineequation based on thecalibration curve of quercetin standard(fig. 3):y=0.0006x, R2=0.9865, where y is an absorbance and x is the mgquercetin g−1.

Total phenol and flavonoid contents of hexaploid cytotypes of both the species of Achyranthesare higher as compared to dodecaploid cytotypes of respective species (table 2).

Fig. 2: Calibration curve for gallic acid

Fig. 3: Calibration curve for quercetin

Table 2:Total phenol and flavonoid content in leaf extract of hexaploid and dodecaploid cytotypes of A. aspera Linn. and A. bidentataBlume

Species Cytotypes Total phenol content (mg/g) Total flavonoidcontent (mg/g)
A. aspera Linn. Hexaploid 9.16±0.84 78.36±1.63
Dodecaploid 7.86±0.08 70.20±1.81
A. bidentata Blume Hexaploid 11.93±0.59 115.92±1.32
Dodecaploid 9.46±0.75 107.76±0.94

Data expressed as mean±SD [n=3]

Phenolic compounds,broadly distributed in the plant kingdom, are a major class of bioactive components. Phenols and flavonoids have drawn increasing attention due to their marked effects in the prevention of various diseases as these possess diverse biological activities,for instance, antioxidant [43], antitumor [44], antiulcer [45],anti-inflammatory [43, 46] and antidepressant activities [47, 48].

Earlier, different leaf extracts of Achyranthes have been evaluated for different biological activities includingantimicrobialactivity [49, 50],anti-inflammatory activity[51], wound healing activity [52], anti-depressant effect [53], cancer chemo preventive activity[54], antinociceptive activity [55], analgesic activity[56], antifertility activities [57], anti-oxidant activity [58] and diuretic activity [59].

Screening of different phytochemical components from leaf extract has also been done earlier [50, 60-62]. However, no report till date is available on the characterization of phenolic constituents in the leaves of A. asperaand A. bidentata having different ploidy level. The present attempt has been carried out to find out the variation in a number of phenolic constituents with variation in ploidy of the plant.In the present investigation, it has been observed that the plants of Achyranthes aspera and Achyranthesbidentatawith the lower ploidy level i.e. hexaploids possess higher amount phenol and flavonoid constituents in the methanol extract of leaves as compared to its dodecaploid counterparts.

Further, RP-HPLC analysis was performed with the purpose of identifying and quantifying one of the phenolic compound i.e. gallic acid. Identification was performed by comparison of their retention’s time and UV absorption spectrum with those of the standards.Five-point calibration curve of astandard sample of gallic acid is given in fig. 4. Fig. 5 shows the chromatograms obtained from theRP-HPLC separation of gallic acid with methanol extracts of different studied cytotypes of A. asperaand A. bidentata. Results revealed a considerable phytochemical (gallic acid) variation in the studied cytotypes of A. asperaand A. bidentata(table 3). Higher concentration of gallic acid is present in dodecaploid plants of A. aspera and A. bidentata as compared to hexaploid counterparts.

Fig.4: Calibration curve for gallic acid

Fig.5: RP-HPLC chromatograms of methanol extracts of leaves of A.Hexaploid cytotype of Achyranthes asperaLinn., B. Dodecaploid cytotype of Achyranthes asperaLinn., C. Hexaploid cytotype of Achyranthes bidentataBlume, D. Dodecaploid cytotype of Achyranthes bidentataBlume. Chromatograms show gallic acid

Table 3: Gallic acid content in the leaf extract of hexaploid and dodecaploid cytotypes of A. aspera Linn. and A. bidentataBlume

Species Cytotypes Gallic acid (mg/g)
A. aspera Linn. Hexaploid 1.01±0.01
Dodecaploid 1.04±0.02
A. bidentataBlume Hexaploid 1.22±0.05
Dodecaploid 1.34±0.03

Data expressed as mean±SD [n=3]

Gallic acid is a poly phenolic compound and chemically known as 3, 4, 5-Trihydroxybenzoic acid. It is extensively used as adietary herbal supplement as occurs in many plant materials in the form of esters, free acids, catechin derivatives and hydrolysable tannins. Gallic acid is evaluated for various biological activities including antioxidant, antimicrobial, anticancer, cardioprotective and neuroprotective [63-67].In addition, no report till date is reported for the determination of gallic acid in different cytotypes of A. asperaand A. bidentata.

CONCLUSION

Present investigation revealed that A. aspera Linn. and A. bidentataBlumeplants show significant intraspecific variability in their total phenol,total flavonoid and gallic acid contentswhich may be accredited to thegenotype of these species. The variation observed in presently studiedbioactive constituents could be considered as an imperative factor for herbal formulation as theefficiency of the herbal remedy may be subjective to the type of genotype used for its preparation. Additionally, it recommends that there is arequirement for the selection of superior genotypes of these species of Achyranthes before recommending the same for therapeutic purpose and conservation.

ACKNOWLEDGMENT

The authors are grateful to the University Grants Commission, New Delhi for providing facilities under the DRS SAP III and ASISTprogramme. Financial assistance to one of us (Lovleen) by Department of Science and Technology, Government of India, New Delhi, under WOS-A is gratefully acknowledged. Thanks are also due to the Head, Department of Botany, Punjabi University, Patiala for necessary laboratory facilities.

AUTHORS CONTRIBUTION

Saggoo MIS provided guidance and critical review. Lovleen researched and wrote this manuscript. Both authors read and approved the final version of this study.

CONFLICT OF INTERESTS

There is no conflict of interest among the authors

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