Document Type : Original Article

Authors

1 Department of Chemistry, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar-263145, U.S. Nagar, Uttarakhand, India

2 Department of Entomology, College of Agriculture, G.B. Pant University of Agriculture and Technology, Pantnagar-263145, U.S. Nagar, Uttarakhand, India

3 Department of Biological Sciences, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar-263145, U.S. Nagar, Uttarakhand, India

Abstract

The objectives of this research were to investigate the qualitative and quantitative analysis of Ethyl Acetate Root Extract of Ardisia solanacea Roxb. (EREAS) and estimation of its biological activities. Phytochemical screening of EREAS showed the abundance of total phenolics, flavanoids, ortho-dihydric phenols, alkaloids, diterpenes and triterpenes etc. The quantitative analysis of EREAS was also carried out by GC/MS and α-amyrenone (13.3%) was found to be the major component. Antifeeding activity monitored through no choice leaf dip method against Spilosoma oblique. The results revealed dose and time dependent antifeeding activity, where the 100% mortality was observed signifying the intense insecticidal activity. The herbicidal activity of extracts was evaluated against the Raphanus raphanistrum seeds. Accordingly, EREAS showed effective herbicidal activity in terms of inhibition of seed germination, coleoptile length and the radical length. Evaluation of antioxidant activity was performed via DPPH radical scavenging, reducing power and metal chelating of Fe2+ activities. EREAS possessed potential antioxidant property and revealed good anti-inflammatory activity.

Graphical Abstract

Chemical composition, insect antifeeding, insecticidal, herbicidal, antioxidant and anti-inflammatory potential of Ardisia solonaceae Roxb. root extract

Keywords

Chemical composition, insect antifeeding, insecticidal, herbicidal, antioxidant

andanti-inflammatorypotentialofArdisiasolonaceaeRoxb.rootextract

BAHAAR ANJUM1, RAVENDRA KUMAR1*, RANDEEP KUMAR1, OM PRAKASH1, R.M. SRIVASTAVA2, D.S. RAWAT3

AND A.K. PANT1

1Department of Chemistry, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar-263145,

U.S. Nagar, Uttarakhand, India

2Department of Entomology, College of Agriculture, G.B. Pant University of Agriculture and Technology, Pantnagar-263145, U.S. Nagar,

Uttarakhand, India

3Department of Biological Sciences, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology,

   Pantnagar-263145, U.S. Nagar, Uttarakhand, India

ABSTRACT

The objectives of this research were to investigate the qualitative and quantitative analysis of ethyl acetate root extract of Ardisia solanacea

Roxb. (EREAS) and estimation of its biological activities. Phytochemical screening of EREAS showed the abundance of total phenolics,

flavanoids, ortho-dihydric phenols, alkaloids, diterpenes and triterpenes etc. The quantitative analysis of EREAS was also carried out by

GC/MS and α-amyrenone (13.3%) was found to be the major component. Antifeeding activity monitored through no choice leaf dip method

against Spilosoma obliqua. The results revealed dose and time dependent antifeeding activity, where the 100% mortality was observed

signifying the intense insecticidal activity. The herbicidal activity of extracts was evaluated against the Raphanus raphanistrum seeds.

    Accordingly, EREAS showed effective herbicidal activity in terms of inhibition of seed germination, coleoptile length and the radical length.

Evaluation of antioxidant activity was performed via DPPH radical scavenging, reducing power and metal chelating of Fe2+ activities. EREAS

possessed potential antioxidant property and revealed good anti-inflammatory activity.

KEYWORDS:

α-Amyrenone

Antifeeding

Anti-inflammatory activity

Antioxidant

Ardisia solanacea

Herbicidal

Insecticidal activity

 

Corresponding author: ravichemistry.kumar@gmail.com

 

1. Introduction

Medicinal plants are sprinkled all over the world, having a large treasure of potential biological active components

hidden within them. It has been reported from time immemorial that plants possess huge medicinal importance and

the human race had a dependence on plant derived components and herbs for their food and health care issues

(Mohammadhosseini et al., 2017; Mohammadhosseini et al., 2019). As per WHO estimates, plant drugs provide nearly

80% to the health needs of the entire world population. Medicinal herbs have been used in the traditional medicine

system for several herbal preparations since prehistoric times (Mohammadhosseini, 2017; Wansi et al., 2018). Many

studies have revealed that plants promote health and well-being to human beings. The utility of herbal remedies is

not only cost-effective but also safe and almost independent from any serious adverse effect. The rural elders, farmers

and tribal cultures have incredible knowledge about the plants being used for various purposes of health since

thousands of years and are still a part of medical practices by folks of various regions of Indian sub-continents and

China, Middle East and African countries, South American and other developing countries of the world (Venditti et al.,

2018). India is a large hub of medicinal plants where more than 45,000 plant species have been documented and

among these, several thousands have been claimed to prove medicinal properties (Mohammadhosseini, 2017).

Ayurveda and other oriental medicinal systems describe how to use the plants in the treatment of a variety of ailments.

To date, various secondary plant metabolites possessing established biological activities have been identified and

discovered. In fact, the plant products are extensively used in various medicinal systems by different practitioners who

require proper documentation and in some reports for further enhancement of their medicinal values

 (Mohammadhosseini, 2017). In morden era, the interest in the area of natural product medicines is growing

exponentially due to the increased awareness of people towards the adverse effects of synthetic drugs (Venditti et al.,

2018).

Family Myrsinaceae R.Br. (Order-Myrsinales) comprises of one of the largest family of flowering plants which includes

about 35 genera and even up to 1000 species (Hutchinson, 1973). The genus Ardisia Sw. has been known to be the

largest genus in the family Myrsinaceae, and even more than 500 species are common in subtropical and tropical

areas globally including Africa, Asia, Australia and America (Chen and Pipoly, 1996). The genus has been recognized

for a number of biological activities like anthelmintic, antimicrobial (Mohammad et al., 2015), analgesic (Shah et al.,

2011), anticonvulsant, anti-inflamatory (Yang et al., 2001), antioxidant, antitumor (Newell, 2010; Ramirez-Mares et al.,

2010), antiplatelet, cytotoxic, trypanocidals (Fujioka et al., 1988; Jia et al., 1994), antifungal, piscicidal and insecticidal

activities (Berenbaum, 1995; Kessler and Baldwin, 2002). A large number of species of the genus Ardisia are used in

the traditional medicine system for different ailments like to cure stomachache and it has a folkloric history in the

treatment of various diseases, such as inflammation, fever, intestinal worms, rheumatism, scabies, snake-bites, cholera,

and cancer treatment (Hideka and De-Mejia, 2005).

ArdisiasolanaceaRoxb. commonly known as Adavimayuri/shoebutton is an important shrub belonging to the family

Myrsinaceae. This plant is native of Pakistan, India, Sri Lanka, China and is widely distributed in Southeast Asia (Bailey,

1925). It is used for a variety of medicinal purposes, e.g. fever, dysmenorrhea, liver disorders, diarrhea, pains, bacterial

infections, rheumatic arthritis, gout, mental disorder, skin sore and vertigo (Khatun et al., 2013). Its roots have been

found to possess potentially active antibacterial activity, cytotoxic, thrombolytic, antioxidant, antirheumatic,

 

februifuge, antidiarrhoic, skin sore and vertigo, while its seed paste has anti-fungal action (Karuppusamy, 2007). The

traditional uses of A. solanacea in various states of India are listed in Table 1. Basha et al. (2016) have reported a

preliminary phytochemical analysis of petroleum ether, ethyl acetate, chloroform, methanol and aqueous root extracts

  • of A. solanacea and showed the presence of flavonoids, saponin, tannins, alkaloids and glycosides. Bauerenol, α-

amyrin, β-amyrin, β-sitosterol, tannin, β-carotene, tocopherol, phenols, phytic acid, lectins and ascorbic acid were

recorded in hexane, benzene and aqueous leaf extracts of A. solanacea (Ahmad et al., 1977; Khan and Ashraf, 1991;

Chandran et al., 2015). In continuation of research work on A. solanacea in our laboratory, qualitative and quantitative

analysis and different biological activities of the ethyl acetate leaf and stem extract of A. solanacea Roxb. have already

been reported by Anjum et al. (2019).

The present research reports a systematic study on both qualitative estimation of secondary metabolites and

quantitative identification of chemical composition (GC/MS), total phenolics estimation, in vitro antioxidant, anti-

inflammatory, insect antifeeding and insecticidal activity of the ethyl acetate root extract of A. solanacea.

2. Experimental

2.1. Collection of plant material

The plant material was collected in blooming phase in June and July 2018 from Kashipur (29° 12' 37.5156'' N and 78°

57' 42.5880'' E.), Tarai region of Uttarakhand, India (Fig. 1). The plant identification was done by one of the co-authors

(D.S. Rawat). The voucher specimen of Ardisia solanacea Roxb. with Acc No. of GBPUH-984/4.4.2019 was deposited

at the herbarium of Department of Biological Sciences, C.B.S.&H., G. B. Pant University of Agriculture & Technology,

Pantnagar.

2.2. Preparation of extract

The fresh root material of A. solanacea was extracted in ethyl acetate using Soxhlet method. The obtained extract was

filtered and concentrated using rotary evaporator. The mean yield of the prepared extract was found to be 0.06%

(w/w) (2.38 g). It was stored at 4 °C for further chemical analysis and determination of biological activity.

2.3. Qualitative analysis

The freshly prepared ethyl acetate root extract of A. solanacea (EREAS) was subjected to qualitative chemical

examination to identify various classes of biologically active constituents using the standard protocols reported in in

literature (Shail, 2011).

2.4. Quantitative analysis

2.4.1. GC/MS analysis

The phytochemicals of the root extract of A. solanacea were identified by using GC/MS. The GC/MS analyses were

carried out using GC/MS-QP 2010 Plus equipment fitted with Ultra Rtx-5MS column of size 30 m × 0.25 mm and film

thickness 0.25 µm. The initial column temperature was programmed to 50 °C for 2 min, and gradually increased to

250 °C at a rate of 3 °C/min. And finally at the rate of 5 °C/min until reaching the final temperature of 310 °C. The

temperature for injector and the detector was kept at 300 °C (split), and 310 °C, respectively. The helium (He) was the

carrier gas used at a linear flow rate of 1.21 mL/min and 69.0 kPa pressure. The operation of MS detector was

performed under EI condition (ionization energy of 70 eV) with an injection volume of 0.1 µL and split mode of 1:120.

 

All compounds of extracts were identified via mass spectral database search (NIST14.lib, FFNSC2.lib, WILEY8.LIB)

followed by the matching of spectra and K.I. (Kovats index) with literature data (Adams, 2007).

2.4.2. Total phenolics, flavonoid and ortho-dihydric phenols content

2.4.2.1. Total phenolic content

Total phenolic content of extract was evaluated using the Folin-Ciocalteu method as per developed protocols

(Singleton and Rossi, 1965). Briefly, 1.0 mL of Folin-Ciocalteu phenol reagent (Sigma-aldrich) was mixed with 0.5 mL of

extract solutions, 1.0 mL of aqueous solution of Na2CO3 (7.0%) and 5.0 mL of distilled water and the mixture was

blended thoroughly. Thereafter, the mixture was kept in the dark for 30 min at 25 °C and the relevant absorbance was

measured at 765 nm. Evaluation of total phenolic content was assessed using the extrapolation of the calibration curve

from the standard gallic acid concentrations measurement. The tests were carried out in triplicate and presented as

gallic acid equivalent (GAE) in mg/g of dry weight.

2.4.2.2. Total flavonoids content

Total flavonoids content of the extract was evaluated using the method developed by Choi et al. (2006). In brief, 1.0

mL of extract was added to 1.25 mL of distilled water and 75 µL of NaNO3 (5.0%) and incubated for 5 min. Then, 150

µL of AlCl3 (10.0%), 500 µL of NaOH (1.0 M) and 275 µL of distilled water were added to the reaction mixture and

mixed well. The absorbance was finally taken at 510 nm. This experiment was carried out in triplicate and total

flavonoid content was expressed as catechin equivalents (CNE) in mg/g of dry weight.

2.4.2.3. Total ortho-dihydric phenols content

Ortho-dihydric phenols content of extract was described by the method of Mahadevan and Sridhar (1986).

Accordingly, to 1 mL of the extract solution, equal volume of HCl (0.5 N), 1 mL of Arnow’s reagent, 2 mL of NaOH (1.0

N) and 4.5 mL of distilled water were also added. The mixture was mixed well and its absorbance was measured at

515 nm. The standard curve was prepared by employing various concentrations of catechol. Total ortho-dihydric

phenols content was expressed with catechol equivalent (CLE) in mg/g of dry weight.

2.5. Antifeedant and insecticidal activity

2.5.1. Test insect

The antifeedant and insecticidal activity of the root extract were evaluated against Bihar hairy caterpillar (Spilosoma

  • obliqua belonging to family Erebidae and order Lepidoptera). Its third instars onward stages cause main harm and

damage to crops by defoliation of the leaf (polyphagous in nature) (Hussain and Begum, 1995; Gupta and

Bhattacharya, 2008; Warad and Kalleshwaraswamy, 2017).

2.5.2. Collection of larvae and maintainance

Insects for test were collected from the field of soybean (Glycine max) crop from C.R.C. (Crop Research Center),

G.B.P.U.A.&T., Pantnagar, Uttarakhand, India in July and August 2018. The rearing of test insects was carried in a glass

jar covered with muslin cloth in the fine laboratory conditions, maintaining temperature at 27 °C and relative humidity

at 75-80%. Test insects were fed with fresh leaf of soybean on every day. And finally the fourth instars larvae were

kept for 12-24 hrs starvation and then employed for their antifeeding potential and insecticidal activity.

2.5.3. Antifeeding activity

 

The antifeeding activity of the root extract of A. solanacea was estimated by employing the developed standard

protocol (Vattikonda and Sangam, 2016) against fourth instar larvae of S. obliqua [Bihar hairy caterpillar (B.H.C.)] using

a disc in no choice bioassay method. The experiment was fulfilled in petri dishes and a moist sheet of filter paper was

placed at the bottom of each petri plate to facilitate relative humidity and also to keep the soybean leaf fresh. The leaf

(each of area 25 sq.cm) were dipped in extract of varying amounts 5%, 10%, 15%, 20% and 25%, dried in air by fanning

and finally introduced in petri plates as food stuff for test insects. To each petri plate, a fourth instar larvae having

starvation of 24 hrs were released. Observations were noted at the time interval of 12, 24, 36 and 48 hrs after the

release of the test insect. The percent antifeeding (feeding inhibition) activity of the plant extract of A. solanacea was

calculated by employing the formula (Eqn. 1):

 

 

 

 

 


Percent antifeeding= Leaf area consumed in control - leaf area consumed in treatment ×100 Leaf area consumed in control + leaf area consumed in treatment

2.5.4. Insecticidal activity


 

(Eqn. 1)

 

The evaluation of insecticidal bioassay of ethyl acetate extract of A. solanacea was conducted by using leaf dip method

(Tabashnik and Cushing, 1987). Soybean leaf was first cleaned and washed with distilled water and air dried for an

hour. Each soybean leaf was cut into an area of 25 sq.cm and then dipped in the solution of extract made in dimethyl

sulphoxide (DMSO) and methanol to smooth the progress of uniform treatment of active ingredient for few seconds.

The obtained leaf discs were kept slanting for 2-3 min on a blotting paper and placed in the tray to drain out excess

solution for two hrs at room temperature. Five, third instar adult larvae, which were starved for 6 hrs, released on

each petri dish in individual petri plate. At the bottom of each plate, blotting paper was placed. These petri plates

were taken under observation for 72 h to watch any insecticidal action. This activity was held under fine laboratory

conditions, maintaining temperature at 27 °C and relative humidity at 75-80%. The mortality (%) was calculated after

24, 48 and 72 h of the treatment using Abbott’s formula (Shen and Shao, 2005). The LD50 values were analyzed by

Probit analysis (Eloff, 1998).

2.6. Herbicidal activity

The seed germination inhibition activity of EREAS under examination was carried on Raphanus raphanistrum (raddish)

seeds by using the method given by Sahu and Devkota (2013) with slight modification.

2.6.1. Source of Raphanus raphanistrum seeds and pendimethalin

Raphanus raphanistrum subsp. sativus (L.) Domin (Syn. Raphanus sativus L.) (Radish) seeds were commercially bought

from the seed store of Pantnagar. In addition, pendimethalin being used as a standard herbicide was supplied by

Weed Control Laboratory, C.R.C, G.B.P.U.A.&T. Pantnagar.

2.6.2. Preparation of test solutions

A stock solution of EREAS (10%) was prepared in distilled water. This stock solution was further serial diluted to four

concentrations of 1%, 2.5%, and 5% and 7.5% respectively for testing seed germination inhibition activity.

Pendimethalin was taken as standard.

2.6.3. Bioassay

 

Raphanus raphanistrum seeds were sterilized using 5.0% sodium hypochlorite solution diluted upto 1:100 and used 15 minutes before experimentation.

The petri plates were firstly covered with ordinary filter papers and then test solution was poured. Ten sterilized

seeds were put in each petri plates and allowed to germinate at (25 ± 1 °C) in the incubator with around 12 hrs of

photoperiod. Distilled water was used as control. The entire experiment was carried out in triplicate. After 90 hrs, the

number of germinated seeds was counted for each amount and inhibition values (%) for seed germination were

calculated.

2.7. In vitro antioxidant activity

2.7.1. DPPH free radical scavenging activity

In vitro evaluation of the free radical scavenging activity of the extracts was performed using the stable radical, 2,2-

diphenyl-2-picrylhydrazyl (DPPH) assay following the method described by Kumar et al. (2019) with little modifications

based on the ability of sample to neutralize DPPH radical. Under the experimental conditions, DPPH is a stable free

radical that can accept hydrogen radical or an electron to convert it into a stable diamagnetic molecule. Various

concentrations of plant extract (50-250 µg/mL) were mixed with 5 mL of methanol solution of DPPH (0.004%), shaken

well and kept in the dark for 30 min at 25 °C for incubation. The absorbance was then measured at 517 nm. DPPH in

methanol without extract was used as negative control. On the other hand, standard antioxidant butylhydroxytoluene

(BHT) was used for positive control. The DPPH radical inhibition (IC%) was calculated by using the Eqn. 2.

Percent inhibition (%) of radical scavenger = IC% = (A₀ - At)/A₀×100 (Eqn. 2)

Where A0, At and IC(%) respectively imply the absorbance value of control sample, the absorbance value of test sample

and the inhibitory concentration(%).

Percent inhibition was plotted against concentrations and the standard curve was drawn using standard antioxidant

(BHT) to calculate the IC50 values for standard and different concentration of extract. A lower IC50 value indicated more

DPPH radical scavenging activity.

2.7.2. Reducing power activity

The reducing power of the A. solanacea extract was determined using the method of Kumar et al. (2019). Different

concentrations of extract (50-250 µg/mL) were mixed with 2.5 mL of phosphate buffer (20 mM, pH 6.6) and 2.5 mL of

potassium ferricyanide [K3Fe(CN)6] (0.5 mL, 1.0%). The mixture was then incubated at 50 °C for 20 min. A portion of

trichloroacetic acid (2.5 mL, 10%) was added to the mixture, which was then centrifuged for 10 min at 650 rpm. 1 mL

  • of the upper layer of the solution was mixed with 5 mL of distilled water and 1 mL of FeCl3 (0.1%) for 10 min, and then

the absorbance was measured at 700 nm, with higher absorbance indicating greater reducing power (Kumar et al.,

2019). All the readings were taken as triplicate with respect to catechin which was used as a standard. The reducing

power of samples was calculated using the Eqn. 3 given below:

Percent inhibition (%) of reducing power activity = (A₀- At)/A₀×100 (Eqn. 3)

Where A0 and At respectively show the absorbance value of control and test samples.

Percent inhibition was plotted against concentrations and the standard curve was drawn using standard antioxidant

(catechin) to calculate the RP50 values for standard and different extract samples. The lower RP50 value indicated greater

reducing power ability.

 

2.7.3. Metal chelating activity

The metal chelating activity of Fe2+ of plant extract was measured according to the reported method by Kumar et al.

(2019). Different concentrations of tested extract samples were mixed with 0.1 mL of FeCl2.4H2O (2mM), 0.2 mL of

ferrozine (5 mM) and 4.7 mL of methanol making volume up to 5 mL. The mixture was then then mixed and shaken.

Ferrozine reacted with the divalent iron to form stable magenta complex species that were very soluble in water. The

mixture was incubated for 30 min at 25 °C, and the absorbance of the Fe2+, ferrozine was then measured at 562 nm.

EDTA was used as standard antioxidant. The ability of the extract to chelate ferrous ion was calculated using the

following formula (Eqn. 4):

Percent inhibition (%) of chelating ability = (Ao- At)/A₀×100 (Eqn. 4)

Where A0 and At respectively account for the absorbance value of control and test samples.

The percent inhibition was plotted against concentrations and the standard curve was drawn using standard

antioxidant (EDTA) to calculate the IC50 values for standard and extracts.

2.8. In vitro anti-inflammatory activity

The method developed by Kar et al. (2012) was adopted for the evaluation of in vitro anti-inflammatory activity with

little modifications. Briefly, a sample was prepared consisting of 2 mL of extract at different concentrations (50-250

µg/mL) and 200 µL of fresh albumin protein (100 ppm) and 2.8 mL of phosphate buffered saline (PBS, pH 6.4). Then,

the reaction mixture was made up to 5 mL and kept for incubation at 37 °C for 15 min and heated at 70 °C for 5 min.

Double distilled water was taken as control. After cooling at room, the corresponding absorbance was measured at

660 nm. Diclofenac sodium was taken as positive control at the same concentration. The percentage inhibition of

protein denaturation was evaluated by the Eqn. 5 given below:

Percent inhibition (%) of protein denaturation = (A0- At)/A0 × 100 (Eqn. 5)

Where A0 and At respectively represent the absorbance value of control and test samples.

2.9. Data analysis

Experiments were performed in parallel triplicate and all data were reported as mean ± standard deviation. The mean

values and standard deviation were calculated statistically. Data analyzed with two-way analysis with replication and

found to be significant at p < 0.05. A statistical analysis was performed using the SPSS 16.0 software package. The

mortality (%) was calculated using Abbott’s formula and the LD50 values were analyzed by Probit analysis.

3. Results and Discussion

3.1. Qualitative analysis

Preliminary phytochemical analysis of EREAS showed the abundance of secondary metabolites such as alkaloids,

carbohydrates, resins, diterpenes, triterpenes, fats and oils. Glycosides, phytosterols, and flavonoids were present in

moderate amounts of EREAS. The plant extracts also showed mild results for saponins, phenols, tannins, proteins and

amino acids. However, the current study showed negative result for ninhydrin test for protein and amino acids. The

results have been summarized in Table 2.

The ethyl acetate root extract of A. solanacea demonstrated the presence of alkaloids, carbohydrates, steroids,

diterpenes, triterpenes, tannins, fats and oils as secondary metabolites with potential biological activities. Basha et al.

(2016) reported preliminary phytochemical analysis of petroleum ether, ethyl acetate, chloroform, methanol and

 

aqueous root extract of A. solanacea and found the presence of flavonoids, saponin, tannins, alkaloids and glycosides.

Samal (2013) reported the presence of flavonoids, glycosides and phenolic compounds in the alcoholic leaf extract of

A. solanacea. Desai et al. (1967) reported the absence of saponins in the stems and roots extracts of A. solanacea.

Chandran et al. (2015) have shown that the leaf aqueous extract of A. solanacea exhibited a small amount of tannin,

β-carotene, tocopherol and ascorbic acid. The phenols, phytic acid and lectins were also recorded in aqueous extract

  • of the plant.

3.2. Quantitative analysis

3.2.1. Chemical composition of extract by GC/MS analysis

The analysis of ethyl acetate root extracts of A. solanacea by GC/MS analysis showed fifty-four compounds (peaks) in

the corresponding GC/MS chromatogram (Table 3 and Fig 2), which were identified except (RT = 42.017 min) according

to their retention time on Ultra Rtx-5MS column and retention indices (NIST14.lib, FFNSC2.lib, WILEY8.LIB, Adams,

2007). The extract mainly comprised of hydrocarbons, fatty acids and terpenoids. α-Amyrenone (13.3%) was found to

be the major component of root extract followed by 4,6,6-trimethyl-2-(3-methylbuta-1,3-dienyl)-3-oxatricyclo

[5.1.0.0(2,4)]octane (10.0%), 3-hydroxy-3,7,11,15-tetramethylhexadecanoic acid silylat (5.4%) and palmitic acid (3.0%).

The other major phytoconstituents investigated from the extract were 1-naphthelenol 5,6,7,8-tetrahydro-2,5-

dimethyl-8-(-1-methylethyl) (1.2%) and docosane (1.0%). However, the minor components in the extract of root

contributing less than 1.0% to the total extract were β-asarone (0.6%), lithocholic acid (0.2%), τ-muurolol (0.1%)

fumaric acid (0.1%), neophytadiene (0.5%), myristic acid (0.1%), estradiol, 3-deoxy (0.4%), cis,cis-linoleic acid (0.3%),

cis-vaccenic acid (0.6%), phytol (0.2%), squalene (0.5%), β-stigmasterol (0.6%), and gorgost-5-en-3-ol, (3.beta.)-, tms

derivative (0.2%). The structures of major compounds present in roots extract are being illustrated in Fig. 3. In our

study, GC/MS analysis of phytochemical of root extract revealed the presence of α and β-amyrin type pentacyclic

triterpenoid compounds, α-amyrenone, 4,6,6-trimethyl-2-(3-methylbuta-1,3-dienyl)-3-oxatricyclo octane, etc. The

presence of these compounds might be responsible for the reported biological activities.

3.2.2. Total phenols, flavonoids and ortho-dihydric phenol content

The total phenolic contents were determined and expressed in gallic acid equivalent (mg of GAE/g of samples), while total

flavonoid content was expressed as catechin equivalents (CNE) in mg/g of dry weight and total ortho-dihydric phenols

content was expressed with catechol equivalent (CLE) in mg/g of dry weight. EREAS contained 323.99 ± 0.04 mg/g GAE

total phenolics, 14.69 ± 0.15 mg/g CNE total flavonoid and 35.55 ± 0.11 mg/g CLE total ortho-dihydric phenol (Table 4).

A. solanacea ethyl aceate root extract exhibited significant phenolic, flavonoid and ortho-dihydric phenol contents.

Amin et al. (2015) reported total phenolic content of A. solanacea MeOH, petroleum ether, CCl4 and CHCl3 extracts

and showed total phenolic content of 58.35 μg of GAE/mg, 69.41 μg of GAE/mg, 37.41 μg of GAE/1 mg and 10.82 μg

  • of GAE/mg of the extracts, respectively. The total phenolic content of aqueous and MeOH extracts of A. solanacea leaf

were 0.030 ± 0.01 and 0.040 ± 0.22 mg of catechin equivalent/mg dried extract, respectively. The flavonoid content

in aqueous and MeOH extract of the plant were 0.257 ± 0.02 and 0.404 ± 0.03 mg of catechin equivalent/mg dried

extract, respectively. Chandran et al. (2015) also reported that total phenolic content present in A. solancea leaf in

significant quantity (113.42 μg/g).

 

3.3. Antifeeding activity

The antifeeding activity screening of the ethyl acetate root extract of A. solanacea (Bihar Hairy Caterpillar) was assessed

against S. obliqua (B.H.C.) using no choice/non preferential leaf dip method (Vattikonda and Sangam, 2016). The higher

antifeeding index indicates decreased rate of feeding by the insect. The result suggests that the extract possesses

significant capability to inhibit the feeding activity of the test insect at all the tested amounts and at all the time

intervals and the results analyzed to be significant (p < 0.05) for all the replications (Table 5).

3.4. Insecticidal activity

EREAS was evaluated for its insecticidal activity against S. obliqua insect using leaf dip bioassay method. Five, third

instar larvae of S. obliqua were used for different concentration of extract to test the bioactivity. EREAS was the most

effective and showed excellent mortality in concentration dependent manner. The extract showed significant mortality

rate of 20.00%, 46.67%, 66.67 and 73.33% at 10, 15, 20, and 25% concentrations respectively after 72 h (Table 6). χ2-

Values, regression equations and LD50 of ethyl acetate root extract of A. solanacea against Bihar hairy caterpillar

(Spilosoma obliqua) insect after 24, 48 and 72 h of treatment have been shown in Table 7.

α-Amyrenone has been reported with remarkable anti-inflammatory and anti-hypersensitivity effects (Rosilene et al.,

2017). The antifeeding activity in the extract was found to be the highest at 25% and sequentially decreased upto

lowest antifeeding activity at 5% in concentration dependent manner. The antifeeding activity is found to be the

highest after 12 h interval of treatment application and decreased with the subsequent time intervals as represented

in Table 5. Crude extracts of various plant species constituting active compounds like sesquiterpenes, diterpenoids,

triterpenes, lactones, quinolene, phenolics, fatty acids, saponins, alkaloids, exhibited antifeeding, insecticidal and

growth inhibitory properties (Arivoli and Tennyson, 2013). Therefore, the antifeeding activity of EREAS could be due

to the major or minor constituents of the extract.

Since promising insecticidal activity was found in the root extract, the actual biocomponent responsible for it should

be further investigated. The mortality percentage was established to be directly proportional to the concentration of

the extract (Table 6). Results of probit analysis for evaluation of the LC50 values, 95% confidence limits and regression

equations at 24, 48 and 72 h for mortality of BHC are presented in Table 7. The LC50 values of the root extracts of A.

solanacea (EREAS) at 24, 48 and 72 h after treatment are 12.02, 10.21 and 8.60 μg/cm2, respectively. The chi-square

values of the root extract at 24, 48 and 72 h after treatment were found to be 0.20, 0.55 and 1.50, as well (Table 7).

3.5. Herbicidal activity

The herbicidal activity of EREAS was assessed against the radish Raphanus raphanistrum seeds to evaluate the

potential impacts of various extracts on different growth parameters such as percent inhibition of seed germination,

percent inhibition of coleoptiles and radical growth.

3.5.1. Inhibition of seed germination

Inhibition of seed germination was estimated as the degree of herbicidal potential. Number of seeds germinated was

firstly counted and then consequently the percent inhibition of the seeds germinated was calculated on day to day

basis till the 100% seed germination was achieved at different concentrations ranges from 1%, 2.5%, 5%, 7.5% and

 

10% of extract. It was measured as 75.69%, 84.83%, 91.32%, 98.23% and 100.00% in increasing order of concentrations,

respectively (Table 8). IC50 was calculated as 0.04 ± 0.02% (Table 9).

3.5.2. Inhibition of coleoptile growth

The percent inhibition of coleoptile growth of the seeds germinated was measured at the time when 100% seed

germination was achieved at different concentrations range from 1%, 2.5%, 5%, 7.5% and 10% of extract. It was

measured as 78.22%, 88.77%, 93.35%, 97.45% and 100.00% in increasing order of concentrations, respectively (Table

10). IC50 was calculated 0.05±0.02% (Table 11).

3.5.3. Inhibition of radicle growth

The percent inhibition of radicle growth of seeds germinated was calculated at the time when 100% seed germination

was achieved at different concentrations range from 1%, 2.5%, 5%, 7.5% and 10% of extract. The percent inhibition of

radical growth was measured as 75.88%, 84.95%, 95.65%, 98.21% and 100.00% in increasing order of concentrations

respectively in case of EREAS (Table 12). IC50 was calculated as EREAS (0.06 ± 0.03%) (Table 13).

A literature survey revealed that till date no herbicidal activity has been reported for A. solanacea and any other species

  • of Ardisia. In the current investigation, phytotoxic ability of the botanicals might be due to the presence of

phytochemical components in the extracts (Wu et al., 1996). Lu et al. (2011) also stated that the herbicidal or

phytotoxicity appeared may be due to the high phytochemical content in the botanicals viz. phenols, flavonoids,

terpenoids, alkaloids etc. or even may be due to the synergistic interaction of the major and the minor components

present in the botanicals (Tiwari et al., 2006; Park et al., 2008).

3.6. In vitro antioxidant activity

The in vitro antioxidant properties of EREAS was evaluated by 2,2-diphenylpicryl hydrazyl (DPPH) radical scavenging

activity, reducing power activity and chelating activity of Fe2+ ion compared with the standard antioxidant (Table 14).

DPPH radical scavenging activities of root extracts were determined for all the tested samples at selected dose levels

with different concentrations (50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL and 250 µg/mL). A continuous decrease

in the absorbance of DPPH was observed in the presence of antioxidants in the tested sample which correlates with

the free radical scavenging potential of the antioxidant. Root extract was found to possess good DPPH radical

scavenging activity with an IC50 value of 190.78 ± 0.14 µg/mL with respect to the BHT used as standard. A good

reducing power activity was achieved with the extract, having the RP50 value of 277.74 ± 1.09 µg/mL, in comparison to the

catechin taken as standard antioxidant (RP50 = 177.71 ± 1.56 µg/mL). EREAS also showed strong metal chelating effect

with an IC50 of 1.99 ± 0.09 µg/mL with respect to EDTA used as standard (IC50 = 2.85 ± 0.01 µg/mL). Statistical analysis

reported to be significantly different (p < 0.01). IC50 values for antioxidant activity expressed as mean ± standard

deviation taken in triplicates and analyzed to be significantly different (p < 0.01).

Significant antioxidant properties of the extract might be possibly due to the significant amount of phenols, flavonoids

and ortho-dihydric phenols (Liu et al., 2008; Liu et al., 2009; Lu et al., 2011). EREAS was also found to possess significant

antioxidant properties since the ethyl acetate root extract of A. solanacea consisted of complex mixture of numerous

components. Total phenolics, the major and minor components in the ethyl acetate root extract of A. solanacea might

 

be responsible for the antioxidant properties. Overall, the order in which plant possesses the activity is metal chelating

> DPPH radical scavenging > reducing power as lower the IC50 value accounts for higher antioxidant activity.

3.7. In vitro anti-inflammatory activity

EREAS exhibited the potential to inhibit protein denaturation at all tested concentrations (50-250 µg/mL) having

percent inhibition from 35.00 ± 0.89% to 88.42 ± 0.84% (Table 15). Statistical analysis reported to be significantly

different (p < 0.01). The IC50 values for extract was 1.79 ± 0.13 µg/mL and for diclofenac sodium was 2.80 ± 0.07

µg/mL.

The results of the current preliminary study stated that the root extract of A. solanacea possessed discernible in vitro anti-

inflammatory effect against the denaturation of albumin protein. Anti-inflammatory activity shown by other species of

Ardisia like A. cornudentata, A. crispa and A. teysmanniana, A. tinctoria with different extracts and active components

presents in the extracts have been reported to be significantly acute and chronic (Sumino et al., 2001; Yang et al., 2001;

Chang et al., 2011). The anti-inflammatory activity performed by the ethyl acetate extract of A. solanacea might be due

to the presence of major components/mixture of components in the extracts. Further definitive studies are still necessary

to find out the mechanism and other components present in various ethyl acetate extracts of A. solanacea for its anti-

inflammatory actions.

4. Concluding remarks

Present research results suggested a potential antioxidant activity for the ethyl acetate root extract of A. solanacea

(EREAS), supporting their use in the traditional medicine and also indicating the plant to be supplemented as an

important component in food, cosmetic and pharmaceutical industries. In addition, the above botanical may be

utilized as Integrated Pest Management Programme (IPM) of Spilosoma obliqua after repeated field experiments.

Because of higher antifeedant activity against lepidopterous pests, the root extracts of A. solanacea may have good

potential for protecting pulse crops suffering heavy damage due to these pests. This plant product may be exploited

for management of other lepidopterous pests in many crops including storage insect’s pest. Because of its presence

in local areas, proper cultivation practices may be adopted for its production and utilization in Pest Management

Programme. The above botanical opens new perspectives on the application of the root extracts of A. solanacea as

novel botanical herbicides for weed management. It can be employed in a new herbicidal formulation and offers new

strategies and pathways for the biopesticide industry to create eco-friendly alternative to chemical herbicides.

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgement

The authors express special reverence to the Advance Instrumentation Research Facility, Jawaharlal Nehru University,

New Delhi for providing GC-MS facility.

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Table 1

Traditional uses of A. solanacea in various state of India.

 

 

 

 

 

 

 


S.N.

States

Traditional uses

References

Plant part

Uses

1.

Andhra Pradesh

Tender leaves

Used as leafy vegetable

Reddy et al. (2007)

2.

Odisha

Root

Root bark paste mixed with dried flower powder of Kanchan and goat milk (4:3:4) is administered twice a day for 15 days

against asthma

Dhal et al. (2015)

Root bark

Fever

Pattanaik et al. (2008)

3.

Tamil Nadu

Leaves

Used as vegetable

Merlin-Franco and Narasimhan, (2009)

Fruits

Edible, used in fits and to treat eye pain

Plant

Plant used in rituals

4.

Kerala

Bark

The bark of this plant is ground with the fruits of Piper nigrum L. var. nigrum, the tuber of Plumbago zeylanica L, and the bark of Zyzyphus trinervia Roxb. the ground mixture is chewed to cure tooth

ache

Devi Prasad and Shyma, (2013)

5.

Meghalaya

Fruits

Ripe fruits eaten raw

Singh et al. (2012)

6.

Great Nicobar

Island

Fruits

Raw fruits

Elanchezhian et al. (2007)

 

 

Table 2

Qualitative phytochemical screening of ethyl acetate root extract of A. solanacea.

 

S.No.

Phytochemical test

Name of the test

EREAS

1.

Alkaloids

Mayer’s test

+++

Wagner’s test

+++

Hager’s test

+++

2.

Carbohydrates

Mohlisch’s test

+++

Fehling’s test

+++

3.

Glycosides

Borntrager’s test

++

Keller-Killiani test

++

4.

Saponins

Froth test

+

Foam test

+

5.

Phytosterols

Salkowski’s test

+

6.

Fats and oils

Stain test

+++

7.

Resin

Acetone-water test

+

8.

Phenols

Ferric chloride test

+

9.

Tannin

Gelatin test

+

10.

Flavonoids

Alkaline reagent test

+

Lead acetate test

+

11.

Proteins and amino acids

Xanthoprotiec test

++

Ninhydrin test

-

Biuret test

+

12.

Diterpenes

Copper acetate test

+++

13.

Triterpenes

Tshugajeu test

+++

EREAS= Ethyl acetate root extract of A. Solanacea (- =absent, + = mild, ++ = moderate, +++ = abundance)

 

Table 3

Chemical composition of the ethyl acetate root extract of A. solanacea(EREAS).

 

S.N

o.

Name of compounds

Mol.

formula

R.I.

Area %

(amount)

1.

undecane

C11H24

-

0.1

2.

dodecane

C12H26

1115

0.7

3.

undecanol

C11H24O

-

0.2

4.

tetradecanol

C14H30O

-

0.3

5.

hexahydro-2h-pyrido(1,2-a) pyrazin-3(4h)-one

C8H14N2O

1317

0.9

6.

heptadecane

C17H36

-

0.8

7.

4,6,6-trimethyl-2-(3-methylbuta-1,3-dienyl)-3-oxatricyclo

[5.1.0.0(2,4)]octane

C15H22O

1407

10.1

8.

nonadecane

C20H42

-

0.3

9.

2-bromo dodecane

C12H25Br

1446

0.1

10.

1-dodecanol, tms derivative

C15H34OSi

1498

0.1

11.

2,4-ditert -butylphenol

C14H22O

1555

0.4

12.

β-asarone

C12H16O3

1568

0.6

13.

pentadecane

C15H32

-

0.3

14.

τ-muurolol

C15H26O

1580

0.1

15.

lithocholic acid, tms

C30H46O3Si2

-

0.2

16.

undecanoic acid, 11-fluoro-, trimethylsilyl ester

C14H29FO2Si

-

0.1

17.

1-naphthalenol,5,6,7,8-tetrahydro-2,5-dimethyl-8-(1-methylethyl

C15H22O

1757

1.2

18.

myristic acid, tms derivative

C17H36O2Si

1788

0.1

19.

9-octadecene, (e)

C18H36

1818

0.4

20.

methyl palmitate

C17H34O2

1878

0.3

21.

estradiol, 3-deoxy

C18H24O

1949

0.4

22.

9-eicosene, (e)

C20H40

2017

0.3

23.

phytol, tms derivative

C23H48OSi

2086

0.2

24.

palmitic acid, tms derivative

C19H40O2Si

-

3.0

25.

eicosane

C20H42

2000

0.8

26.

methyl linolelaidate

C19H34O2

2093

0.1

27.

heptadecane

C17H36

-

0.1

28.

heneicosane

C21H44

2109

0.9

29.

neophytadiene

C20H38

-

0.5

30.

cis-vaccenic acid

C18H34O2

2175

0.6

31.

3-hydroxy-3,7,11,15-tetramethylhexadecanoic acid, silylat

C26H56O3Si2

-

5.4

32.

cis,cis-linoleic acid

C18H32O2

2183

0.3

33.

stearic acid, tms derivative

C21H44O2Si

2186

0.3

34.

oleic acid, (z)-, tms derivative

C21H42O2Si

2194

0.4

35.

linolsaeue,trimethylsilylester

C21H40O2Si

2202

0.1

36.

fumaric acid, bis (tms)

C10H20O4Si2

-

0.1

37.

2,2,10,10- tetramethyl-5-[(tms)ox)]

C14H36O3Si3

-

0.1

38.

epimethendiol-diotms

C26H48O2Si2

-

0.2

39.

1-heneicosanol

C21H44O

2351

0.2

40.

tetratetracontane

C44H90

-

0.5

 

41.

hexacosane

C26H54

2606

0.3

42.

mevalonic acid, 3tms

C15H36O4Si3

-

0.1

43.

α-amyrenone

C30H48O

-

13.3

44.

β-stigmasterol

C29H48O

2739

0.6

45.

stigmasta-5,22-dien-3-ol

C29H48O

-

0.4

46.

β-sitosterol, tms

C32H58OSi

-

0.2

47.

docosane

C22H46

2200

1.0

48.

gorgost-5-en-3-ol, (3. beta.)- tms

C33H58OSi

2826

0.2

49.

nonadecane

C19H40

-

0.1

50.

isononacosane

C29H60

-

0.2

51.

lupeol

C30H50O

2848

0.1

52.

α-amyrin

C30H50O

2873

10.2

53.

squalene

C30H50

2914

0.5

54.

vitamin E

C29H50O2

3149

0.2

 

Table 4

Quantitative estimation of total phenolics, total flavonoids and ortho-dihydric phenol of ethyl acetate root extract of

A. solanacea (EREAS).

 

Phytochemical assay

EREAS

Total phenolic content

317.93 ± 0.02 mg/gm of GAE

Total flavonoids content

14.69 ± 0.15 mg/gm of CNE

Ortho-dihydric phenol content

35.55 ± 0.11mg/gm of CLE

EREAS-Ethyl Acetate Root Extract of A. solanacea, GAE-Gallic Acid Equivalent, CNE-Catechin Equivalent, CLE-Catechol

Equivalent

 

Table 5

Antifeedant activity (%) of ethyl acetate root extracts A. solanacea (EREAS) with different concentrations.

Doses

%

After 12 h

After 24 h

After 36 h

After 48 h

Leaf area

consumed

Antifeeding

activity(%)

Leaf area

consumed

Antifeeding

activity(%)

Leaf area

consumed

Antifeeding

activity(%)

Leaf area

consumed

Antifeeding

activity(%)

5

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

10

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

15

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

20

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

25

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

00 ± 00

100.00 ± 00

Control

17.35 ± 8.13

-

18.93 ± 5.84

-

20.72±

4.02

-

25.00 ± 00

-

% antifeeding activity of ethyl acetate extract calculated by the method, reported by the earlier Vattikonda and

Sangam, 2016, data analyzed with two-way analysis with replication and found to be significant at p < 0.05.

 

Table 6

Mortality percentage of Bihar hairy caterpillar (Spilosoma obliqua) insect, treated with ethyl acetate root extract of A.

solanacea (EREAS) by leaf dip method.

 

S.No.

Concent ration

No of

insect used

No of insect dead

Average mortality(%)

Corrected mortality(%)

24 h

48 h

72 h

24 h

48 h

72 h

1.

25%

5

3

4

4

60.00

80.00

80.00

73.33 ± 02

2.

20%

5

2

4

4

40.00

80.00

80.00

66.67 ± 03

3.

15%

5

1

3

3

20.00

60.00

60.00

46.67 ± 08

4.

10%

5

0

1

2

0.00

20.00

40.00

20.00 ± 06

5.

5%

5

0

1

2

0.00

20.00

40.00

20.00 ± 04

The % mortality was calculated after 24, 48 and 72 h of the treatment using Abbott’s formula (Shen and Shao, 2005).

 

Table 7

χ2-Values, regression equations and LD50 of ethyl acetate root extract of A. solanacea against Bihar hairy caterpillar

 (Spilosoma obliqua) insect, after 24, 48 and 72 h of treatment.

HAF

LC30

(μg/cm2)

LC 50

(μg/cm2)

LC 90

(μg/cm2)

Fiducial limit

Chi square

Reg equation

Lower

limit

Upper

limit

24

7.20

12.02

42.40

8.01

16.57

0.20

y = 2.332x + 2.485

48

6.05

10.21

36.97

6. 13

13.92

0.55

y = 2.312x + 2.674

72

5.04

8.60

32.21

4.40

11.88

1.50

y = 2.316x + 2.835

The LD50 values were analyzed by Probit analysis (Eloff, 1998)

 

Table 8

Inhibition of seed germination (%) of ethyl acetate root extract of A. solanacea.

 

Extract/standard

% Inhibition of germination

Replicates-3

1.0%

2.5%

5.0%

7.5%

10.0%

EREAS

Mean

68.65

84.65

86.43

96.43

100.00

STD

75.69

84.83

91.32

98.23

100.00

Pendimethalin

Mean

100.00

100.00

100.00

100.00

100.00

STD

00.00

00.00

00.00

00.00

00.00

EREAS= Ethyl Acetate Root Extract of A. solanacea. Data analyzed with two-way analysis with replication and found to

be significant at p < 0.05.

 

Table 9

IC50 values of seed germination inhibition by ethyl acetate root extract of A. solanacea.

 

Extract

IC50 Values

IC50

R1

R2

R3

EREAS

0.06

0.04

0.02

0.04 ± 0.02

EREAS= Ethyl Acetate Root Extract of A. solanacea.Data analyzed with two-way analysis with replication and found to

be significant at p < 0.05.

 

634 Table 10

635 Inhibition of coleoptile growth (%) of ethyl acetate root extract of A. solanacea.

 

Extract/standard

% Inhibition of coleoptiles growth

Replicates-3

1.0%

2.5%

5.0%

7.5%

10.0%

EREAS

Mean

75.69

84.83

91.32

98.23

100.00

STD

6.24

5.94

4.47

1.74

0.00

Pendimethalin

Mean

100.00

100.00

100.00

100.00

100.00

STD

00.00

00.00

00.00

00.00

00.00

636

637 EREAS= Ethyl Acetate Root Extract of A. solanacea. Data analyzed with two-way analysis with replication and found to

638 be significant at p < 0.05.

639

 

640

 

641 Table 11

 

642 IC50 values of coleoptile length germination by ethyl acetate root extract of A. solanacea.

 

Extract

IC50 Values

IC50

R1

R2

R3

EREAS

0.06

0.04

0.02

0.04 ± 0.02

643 EREAS= Ethyl Acetate Root Extract of A. solanacea. Data analyzed with two-way analysis with replication and found to

644 be significant at p < 0.05.

645

646

647

648

649

650

651

652

653

654

655

656

657

 

Table 12

% Inhibition of radical growth of ethyl acetate extract of A. solanacea.

 

Extract/standard

% Inhibition of radical growth

Replicates-3

1.0%

2.5%

5.0%

7.5%

10.0%

EREAS

Mean

75.88

84.95

90.44

97.63

100.00

STD

5.21

4.43

4.53

2.57

00.00

Pendimethalin

Mean

100.00

100.00

100.00

100.00

100.00

STD

00.00

00.00

00.00

00.00

00.00

660 EREAS= Ethyl Acetate Root Extract of A. solanacea. Data analyzed with two-way analysis with replication and found to

661 be significant at p < 0.05.

662

 

663

 

Table 13

 

IC50 values of radical germination inhibition by ethyl acetate root extract of A. solanacea.

 

Extract

IC50 Values

IC50

R1

R2

R3

EREAS

0.06

0.04

0.02

0.04 ± 0.03

EREAS= Ethyl Acetate Root Extract of A. solanacea. Data analyzed with two-way analysis with replication and found to

be significant at p < 0.05.

 

681 Table 14

682 In vitro antioxidant activities of the ethyl acetate root extract of A. solanacea expressed in terms of their IC50 values.

683

Extract/standard

DPPH radical scavenging activity (μg/mL)

Reducing power activity

(μg/mL)

Metal chelating activity

of Fe2+ (μg/mL)

EREAS

190.78 ± 0.14

277.74 ± 1.09

2.79 ± 0.01

BHT

107.48 ± 0.70

-

-

Catechin

-

177.71 ± 1.56

-

EDTA

-

-

2.85 ± 0.01

684 EREAS-Ethyl Acetate Root Extract of A. solanacea, BHT-Butylated Hydroxyl Toluene, DPPH-2,2-Diphenyl-1- 685 picrylhydrazyl, EDTA=Ethylene Diammine Tetraacetate (Na salt), All the IC50 values expressed as mean ± standard 686 deviation taken in triplicates and analyzed to be significantly different (p < 0.01).

687

688

689

 

690 Table 15

691 In vitro anti-inflammatory activity in terms of IC50 values for ethyl acetate root extract of A. solanacea (EREAS).

Extract/standard

IC50 value

EREAS

1.79 ± 0.13

Diclofenac sodium

2.80 ± 0.07

692 % in-vitro anti-inflammatory activity of Ethyl Acetate Root Extract of A. solanacea (EREAS) versus the standard anti- 693 inflammatory agent (diclofenac sodium), IB50values plotted as mean ± standard deviation with percent inhibition at 694 various concentrations are significantly different (p < 0.01).

695

696

697

698

699

700

701

702

703

704

705

706

707

708

709

710

 

711

 

 

 

 

 

 

 

 

 

 

 

 

 

712

713 Fig. 1. Shrub/small tree of Ardisia solanacea Roxb. growing in its natural habitat (Photograph taken from Tarai region,

714 Uttarakhand, India).

715

716

717

718

719

720

721

722

723

724

725

726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

 

744

745

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
   

 


746

747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

763

764

765

766


 

Retention time (min)

Fig. 2. Gas chromatogram of ethyl acetate root extract of A. solanacea (EREAS).

 

 

 

 

α-Amyrenone

 

 

 

 

 

α-Amyrin

 

 

 

 

 

O

 

 

 

4,6,6-Trimethyl-2-(3-methylbuta-1,3-dienyl)-3-oxatricyclo [5.1.0.0(2,4)]octane

 

767 Fig.3.Structure of major compounds present in ethyl acetate root extract of A. solanacea.

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