A Comparative Study of the Antioxidant and ACE Inhibitory Activities of Selected Herbal Extracts

Journal Pre-proof A Comparative Study of the Antioxidant and ACE Inhibitory Activities of Selected Herbal Extracts Neha Chaudhary, Latha Sabikhi, Shaik Abdul Hussain, Sathish Kumar M H

PII:

S2210-8033(20)30015-4

DOI:

https://doi.org/10.1016/j.hermed.2020.100343

Reference:

HERMED 100343

To appear in:

Journal of Herbal Medicine

Received Date:

15 November 2017

Revised Date:

12 November 2018

Accepted Date:

10 February 2020

Please cite this article as: Chaudhary N, Sabikhi L, Hussain SA, M H SK, A Comparative Study of the Antioxidant and ACE Inhibitory Activities of Selected Herbal Extracts, Journal of Herbal Medicine (2020), doi: https://doi.org/10.1016/j.hermed.2020.100343

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A Comparative Study of the Antioxidant and ACE Inhibitory Activities of Selected Herbal Extracts

Neha Chaudhary1* [email protected], Latha Sabikhi2 [email protected], Shaik Abdul Hussain3 [email protected], Sathish Kumar M H4 [email protected] ICAR-NDRI, KARNAL 1

Research Scholar, Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal-132 001, Haryana, India, Contact No: +91- 9467584177 2

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Principal Scientist & Head, Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal-132 001, Haryana, India, Contact No: +91-9896075404 3

Scientist, Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal-132 001, Haryana, India 4

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Corresponding author

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*

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Scientist, Dairy Technology Division, ICAR-National Dairy Research Institute, Adugodi, Banglore-560030, India

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Graphical abstract

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Abstract

Some herbal bioactives possess high antioxidant and angiotensin converting enzymes (ACE)

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inhibition activity, which affects blood pressure levels. Herbal bioactives promote health benefits via different molecular mechanisms and most of these mechanisms are yet to be fully elucidated. In the present study, health promoting potential of aqueous extracts of six herbs

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viz. Allium sativum L., Withania somnifera (L.) Dunal, Ocimum sanctum L., Hibiscus sabdriffa L., Ginkgo biloba L. and Emblica officinalis at different concentrations (50 to 10,000 µg/mL) were assessed through their antioxidant and ACE inhibitory potential. Results

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showed that the DPPH (2, 2-diphenyl-1-picrylhydrazyl) activity of the six different herbs ranged from 1.58±0.63 to 11.39±0.15 mM/mL trolox equivalent and was in the order of

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Emblica officinalis >Withania somnifera >Hibiscus sabdriffa >Ginkgo biloba >Ocimum sanctum =Allium sativum at 1000 µg/mL concentration. Emblica officinalis had IC50 value 180.35±1.17 µg/mL. The corresponding observation for the herb with 2, 2'-azino-bis (3ethylbenzothiazoline-6-sulphonic acid) (ABTS) was 167.19±1.04 µg/mL. Ferric reducing antioxidant power (FRAP) values were significantly higher for Emblica officinalis than the other herbs, and varied from 1.78±0.08 to 251.62±6.31 mM/mL ascorbic acid equivalent at different concentrations (50 to 10000 µg/mL). Emblica officinalis had the strongest ACE

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inhibition potential (82.08±5.18%), followed by Withania somnifera, Hibiscus sabdriffa, Ginkgo biloba, Ocimum sanctum and Allium sativum. ACE inhibitory activity of herbs studied was lower (31.64 ± 5.05%) than that of Captopril (0.002173 µg/mL), a standard medicine used for hypertension. The study compared and elucidated the antioxidant and ACE inhibitory potential of aqueous extracts of herbs at different concentrations. The study results may be useful in the selection of herbal bioactives for functional foods and aqueous nutraceutical formulations.

Abbreviations Used: Inhibitory concentration (IC50); Angiotensin converting enzyme

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(ACE); Radical scavenging activity (RSA); 2,2-diphenyl-1-picrylhydrazyl (DPPH); 2,2'-

Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt (ABTS); 2,4,6-tripyridyl triazine (TPTZ); 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox);

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Hippuryl-His-Leu acetate salt (HHL); Hippuric acid (HA); benzene sulfonyl chloride (BSC).

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Keywords: Herbs; Emblica officinalis; Antioxidant activity; ACE inhibitory activity

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1. Introduction Herbs and herbal medicines have been used for the treatment of diseases in almost all civilisations. Herbal medicines are popular due to their effectiveness, low cost and relatively few side effects. More than 2000 herbs have been listed in traditional herbal medicinal systems for the treatment of hypertension and oxidative stress. These medicines reportedly can be cardioprotective (thus reducing the risk of heart damage), cardioactive (affecting the activity of heart), cardiotonic (increasing the efficiency of the heart muscle) and/or circulatory stimulating activities (Hussain et al., 2015). Garlic (Allium sativum L.), ashwagandha (Withania somnifera (L.) Dunal), tulsi (Ocimum sanctum L.), roselle (Hibiscus

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sabdriffa L.), ginkgo (Ginkgo biloba L.) and Indian gooseberry (Emblica officinalis Gaertn.) are important herbs in the Indian traditional medical system of Ayurveda. These herbs are

very effective in the reduction of hypertension and oxidative stress. It is well documented that tulsi (2 g tulsi powder/kg/day for 30 days in human model), roselle (9.6 mg anthocyanin/day

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for 4 weeks), garlic [960 mg garlic extract (2.4 mg allicin)/kg/day for 12 weeks to human],

ashwagandha (25, 50, 100 mg/kg/day to mice), ginkgo extract (60, 90, 180 mg/kg/day to rat),

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and Indian gooseberry (500mg/kg/day for 42 days in human model) have significantly reduced the blood pressure and promoted vasodilation in studies(Kochhar et al., 2009; Chaudhary et al., 2014; Ojeda et al., 2010; Ried et al., 2010; Mohanty et al., 2004; Liu et al.,

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2007; Mansour et al., 2011). This is due to the presence of biomolecules carotenoids, triterpenes, flavonoids, glycosides, alkaloids, saponins and terpenoids. Some of the mechanisms by which these bioactives may prevent some chronic and cardiovascular

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diseases are through the altering of antioxidant activity. These mechanisms involve superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), glutathione peroxidase

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(GPx), malondialdehyde (MDA) and glutathione reductase (GR), inhibition of angiotensin converting enzyme; exhibiting vasodilatory effect, improving lipid profile parameters by

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modulating low density lipoprotein (LDL), very low density lipoprotein (VLDL), triglycerides (TGs), high density lipoprotein (HDL) and total cholesterol (TC) (Arya and Gupta, 2011).

Reactive oxygen species (ROS) comprise of reactive chemical species with strong

oxidizing tendency and lead to excess free radicals in the body that oxidize low density lipoproteins and cause body degradation over time. The studies Kanyaiya et al. (2014) and Sawale et al. (2013) showed improved immunomodulatory effect, increased antioxidant potential and reduced glutathione level by the administration of Pueraria tuberosa extract in 13

mice using milk as carrier. Bioactives present in herbs act as natural antioxidants which neutralise ROS. They may act independently or in combination as cardioprotective agents. They use different block defence mechanisms to stop the growth of free radical species. Chaphalkar et al. (2017) reported that hydroalcoholic extract of Emblica officinalis bark (dose: 1000 mg/kg, p.o. for 30 days) showed hepatoprotective activity in male Wistar rats. Antioxidant potential of a variety of products can be estimated by different methods such as trolox equivalent antioxidant capacity (TEAC) using 2, 2-azinobis (3ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay as an oxidant, the ferric reducing antioxidant power assay (FRAP), 2,2’-diphenyl-1-picrylhydrazyl (DPPH) free radical

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scavenging potential, oxygen radical absorption capacity (ORAC), total radical absorption potentials (TRAP), and the photochemiluminescence (PCL) assays (Schlesier et al., 2002).

Herbs have a mixture of antioxidant moieties, therefore their assay reaction kinetics may be

more complex than those of standard compounds. ABTS assay reduces the pH from 7.4 to 4.5

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providing a stable reaction medium for natural products containing phenolics and helps in sensitive, long term measurement. The DPPH assay is most suited for samples containing

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lipophilic antioxidants (Ozgen et al., 2006). ABTS assay offers a stable method and is a good choice of combination with FRAP and DPPH methods. Therefore, these three methods were used for the accurate estimation of antioxidant potentials of different herbal extracts (Ozgen

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et al., 2006).

Angiotensin-I-converting enzyme (ACE) also plays an important role in blood

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pressure regulation and cardiovascular function. The enzyme converts the hormone angiotensin I to the active vasoconstrictor angiotensin II. ACE indirectly increases the blood pressure by constricting blood vessels and is also involved in the degradation of bradykinin

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(Delacroix et al., 2014), which is a vasodilator. Various medicinal plants such as Acacia nilotica, Acacia Senegal, Withania somnifera, Ginseng, Ficus hispida, Hibiscus sabdriffa,

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Ginkgo biloba, Ocimum sanctum, Allium sativum and Aloe vera (Basannavar et al., 2014) have antihypertensive and diuretic properties and have been tested for their ACE inhibitory activity. Hence, the current study was undertaken to screen the six herbs (selected on the basis of literature available) for the best cardioprotective actions accounted on the basis of their antioxidant ability and angiotensin converting enzyme (ACE) inhibitory activities. 2. Material and Methods 2.1. Materials

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Six herbal extracts namely garlic, ashwagandha, tulsi, roselle, ginkgo and Indian gooseberry were selected for the study. The extracts were purchased from M/s. Plantae Herbal Extract Pvt. Ltd, New Delhi. All the chemicals used in the study were of analytical grade and purchased from Sigma-Aldrich and Thermo Fischer Scientific, India. 2.2. Methods 2.2.1. Preparation of extracts Dry water- soluble powdered extracts of the above mentioned six herbs were obtained from M/s. Plantae Herbal Extract Pvt. Ltd, New Delhi. Full details of batch number, plant part used, % active compounds and quality control data are shown in Table 1. Using water as

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a solvent, different dilutions (50 to 10000 µg/mL) of the powdered herbal extracts were prepared. Distilled water was used as a common solvent for each herbal extract. 2.2.1. DPPH method

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DPPH assay was performed as previously described by Mimica-Dukic et al. (2004) with some modifications. The samples were prepared in different concentrations with 60%

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methanol (acidified with 0.1% concentrated HCl). To 0.1 mL of sample, 2.9 mL of 0.1 mM DPPH (prepared in 80% methanol) was added and incubated at room temperature for 30 min

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in darkness. Standard solution of trolox was also prepared by using 80% methanol and treated as control. The absorbance was measured at 517 nm using UV-vis Spectrophotometer (GENESYS 10UV, ThermoSpectronic, ThermoElectron Scientific Instruments LLC,

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Madison, WI USA) in darkness. Radical scavenging activity (RSA) in terms of percentage inhibition was calculated by using the following equation.

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𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑏𝑙𝑎𝑛𝑘 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒 /𝐴𝑏𝑙𝑎𝑛𝑘 ) ∗ 100 where, Ablank was absorbance of control and Asample, absorbance of sample. Free radical

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scavenging activity was calculated using the standard curve and expressed as mM/mL trolox equivalent. Inhibition constant (IC50) values, which represented the concentration of active component that resulted in 50% scavenging, were calculated from the plot of inhibition per cent against concentration. 2.2.2. ABTS Assay ABTS assay was carried out as described earlier by Arnao et al. (2001) with some modifications. Initially, 1900 µL of ABTS+ working solution was added to the 100 µL of blank and standard and mixed immediately to start the reaction. After 6 min reaction time the 13

absorbance was measured at 734 nm. For the preparation of standard curve stock solution trolox was diluted with phosphate buffer saline so that the final concentration of the dilution series ranged from 0.025 to 0.450 mM. Antioxidant activity in terms of percentage inhibition was calculated by using the following equation. 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑏𝑙𝑎𝑛𝑘 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒 /𝐴𝑏𝑙𝑎𝑛𝑘 ) ∗ 100 where, Ablank was absorbance of control and Asample, absorbance of sample. For the standard curve, trolox stock solution was diluted with phosphate buffer saline in such a way that the standard curve was linear between 100 and 1000 µmol trolox. Results were expressed as antioxidant activity µmol/ml trolox equivalent.

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2.2.3. FRAP method

The total antioxidant potential of herbs was determined using ferric reducing and

antioxidant power (FRAP) assay (Benzie and Strain, 1996) with some modifications. FRAP reagent was freshly prepared and mixed in the proportion of 10:1:1 (v:v:v) for A:B:C

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solutions [A: 300 mmol/l sodium acetate tri-hydrate in glacial acetic acid buffer (pH = 3.6); B: 10 mmol/l TPTZ in 40 mmol/l HCl; C: 20 mmol/l FeCl3.6H2O]. A 0.1 µL of herbal

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extracts aliquot was taken in microtubes and 3 mL of FRAP reagent was added. The samples were incubated at 37oC in water bath for 30 min in dark and then readings were taken at 593

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nm using spectrophotometer. Ascorbic acid was used as standard and results were expressed in mmol/ml ascorbic acid equivalent.

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2.2.4. Angiotensin Converting Enzyme (ACE) Inhibition Assay ACE inhibitory activity was measured by the procedures given by Cushman and Cheung (1971) with some modifications as per Li et al. (2005). For each assay, a sample

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solution of ACE inhibitor (20 µl) with 50µl of 5mmol HHL in 100 mmol sodium borate buffer (pH 8.3) containing 300 mmol NaCl was pre-incubated at 37oC for 5 min. The reaction

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was initiated by the addition of 10 µl of ACE solution (100 mU/ml), and the mixture was incubated at 37ºC for 30 min. The reaction was stopped by adding 100µl of 1M HCl. Sodium borate buffer was then added to the reaction mixture to a volume of 0.5 mL. Then 600µl quinolone and 200µl of BSC were added to the reaction mixture and incubated at 30oC for 30 min in the dark. After adding 3700µl of ethanol, it was once again incubated at 30oC for 30 min in the dark, followed by measurement of absorbance at 492 nm. The extent of inhibition was calculated as follows:

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𝐵−𝐴 𝐴𝐶𝐸 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟𝑦 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) = ( ) × 100 𝐵−𝐶 Where, B - the absorbance of control (buffer added instead of test sample), C - absorbance of the reaction blank (HCl was added before ACE) and A - absorbance in the presence of the sample. 2.2.5. Total polyphenols and total flavonoids Total polyphenols and total flavonoids were estimated as per the method described by Madaan et al. (2011).

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2.3. Statistical analysis All values are presented as mean±SD. The results were analysed by one-way

ANOVA and two-way ANOVA using IBM SPSS Statistics 20 software, to determine

statistical significance among groups. Significant difference was accepted at p<0.05. IC50

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values were calculated using online software http://www.changbioscience.com. Each analysis was repeated four times to obtain reproducibility and to avoid experimental errors.

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3. Results and Discussion

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3.1. Antioxidant activity

DPPH method is based on the ability of DPPH which is a stable free radical, to decolourize in the presence of antioxidants. Inhibitory concentration (IC50) value is the

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measure of effectiveness of a substance, in inhibiting a specific biochemical function. The free radical scavenging activity and inhibitory concentration (IC50) of the six different herbs at concentrations ranging from 50 to 10000 µg/ml are listed in Table 2. It is evident from the

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results that Emblica officinalis had the highest free radical scavenging activity (mmol/ml Trolox equivalent) among the six herbs. The free radical scavenging activity values of six

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herbs ranged from 1.58±0.63 to 11.39±0.15 mmol/ml trolox equivalent and at 1000 µg/ml concentration in this order of Emblica officinalis >Ashwagandha>Roselle>Ginkgo>Tulsi = Garlic. The free radical scavenging activity of herbs increased with increase in their concentration applied in the assay. The free radical scavenging activity of herbs varied significantly (p<0.05) with change in concentration except for ginkgo, tulsi and garlic. This may be due to difference in the chemical nature of active components as well as steric inaccessibility of the molecules at this concentration (Ahmed et al., 2015). The relative free radical scavenging activity of Emblica officinalis was higher than the remaining herbs at all 13

concentrations tested. The free radical scavenging activity of Emblica officinalis reduced the extract beyond the concentration of 1000µg/ml, which could be ascribed to saturation of reaction mixture due to high amount of antioxidants present in the herb. The IC50 values obtained for each herb ranged from 180.35 to 5068.66 µg/ml trolox equivalent, in the order Indian gooseberry >Garlic>Roselle>Ashwagandha>Ginkgo>Tulsi. The free radical scavenging activity of herbal extracts were dose dependent as shown in the Table 2. The IC50 values of the herbal extracts were 180.35µg/ml, 1362.22µg/ml, 4612.17µg/ml, 1034.87µg/ml, 4950.33µg/ml and 5068.66 µg/ml for Indian gooseberry, Roselle, Ashwagandha, Garlic, Ginkgo and Tulsi, respectively (as provided in Table 2). The reported

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values for Indian gooseberry aqueous extract, air dried immature plant of Garlic and Roselle extract in different solvents were 142.6µg/ml, 1030µg/ml and 289.01-1051.72µg/ml,

respectively as reported by various authors (Liu et al., 2008; Bozin et al., 2008; Yang et al., 2012). The IC50 values of the herbal extracts strongly depends on the method and type of

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solvent used for processing the extract (Alam et al., 2012; Kaur et al., 2012). The limitation of this method is that it is more suitable for the extraction of polar polyphenol compounds

DPPH method (Amorati et al., 2015).

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and flavonoids. It is unfortunate that only methanol and ethanol are suitable solvents for the

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ABTS assay measures the relative capacity of antioxidants to scavenge the ABTS+ radical compared to the antioxidant potency of trolox, used as a standard. The antioxidant activities of the six herbs at the concentration ranging from 50 to 10000 µg/ml are depicted in

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Table 3. It was found that ABTS values increased with an increase in the concentration of the herbal extracts. Emblica officinalis had the highest antioxidant activity among all the herbs at different concentrations. Shukla et al. (2009) reported that the antioxidant activity for the

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aqueous and alcoholic extract of Emblica officinalis was 7.78±0.17 and 6.68±0.11µmol/ml, respectively, at 1000µg/ml, while the present study revealed that antioxidant activity for

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aqueous extract was much higher at 4884.88±11.39µmol/mL trolox equivalent for the same concentration. Khopde et al. (2001) concluded that high antioxidant activity of Emblica officinalis was due to the presence of free radical scavenging compounds. The reason for the variation between both investigations may be the very high amount of polyphenols present in the extracted sample that were used in this study. The inhibitory concentration value of Indian gooseberry was the lowest (167.19µg/ml) followed by Garlic (258.96µg/ml), Roselle (540.88µg/ml), Ashwagandha 13

(541.76µg/ml), Ginkgo (654.61µg/ml) and Tulsi (6649.22µg/ml). This indicates that Indian gooseberry exhibited 50% inhibition at very low concentrations, while in the case of Tulsi, a high concentration of extract was required to inhibit 50% of reactive oxygen species. The effectiveness of polyphenols depends on the molecular weight, number of aromatic rings and nature of hydroxyl group’s substitution than specific functional groups (Hagerman et al., 1998). The major drawback of this method is that the radicals formed are not very stable and results are not reproducible (Amorati et al., 2015). FRAP method involves reduction of ferric to ferrous ion at a low pH that forms a colored complex ferrous-tripyridlytriazine, and is used for the assessment of antioxidant

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power. Emblica officinalis has shown best activity followed by Roselle and Ashwagandha. Garlic, Ginkgo and Tulsi exhibited the least antioxidant power. Emblica officinalis FRAP

value varied from 1.78±0.08 to 251.62±6.31mmol/ml ascorbic acid equivalent at different

concentrations (50 to 10000µg/ml) and were significantly (p<0.05) different from the other

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herbs investigated (Table 4). However, at low concentration, the difference was not

significant, probably because of the non-availability of phenolic hydrogen that reduces the

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FRAP reagent to significant levels. Sharma et al. (2009) who compared the FRAP value of Emblica officinalis reported that it was higher than those of Moringa oleifera and Trichosanthes dioica. Charoenteeraboon et al. (2010) studied the FRAP value of Emblica

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officinalis (7.46±0.56μmol FeSO4/mg) and concluded that the reduction of ferric ions to ferrous sulfate by the extract was dose-dependent.

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In this study, lower FRAP values were obtained for garlic and tulsi when compared to the results of Nencini et al. (2011), and Wangcharoen and Morasuk (2007). This variation may be attributed to the differences in the sample type and composition used in these studies.

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Reducing activity of ashwagandha leaf fractions in methanolic extract reported by Alam et al. (2012) were found to be lower than findings of the present study. This may be due to the

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difference in solvent used for extraction of active constituents from the herbs. The antioxidant activity of herbal extracts depends on the chemical nature of its constituents. Hence, the results of the current study differ from Alam et al. (2012). The limitation of FRAP method is that it is non-specific and arbitrarily depends on the reaction time (Amorati et al., 2015). 3.2. ACE inhibitory activity The ACE inhibitory percentage of captopril was 31.64% at 0.1µmol concentration. All herbal extracts were estimated at the dose of 10mg/ml (Table 5). ACE activity for 13

Emblica officinalis was 82.08%, highest among all herbal extracts, followed by ashwagandha (62.35%), roselle (58.89%), garlic (39.57%), tulsi (22.87%) and ginkgo (11.21%). The inhibitory activity of Emblica officinalis was significantly (p<0.05) different from the rest of the herbs except ashwagandha. The ACE activity of Emblica officinalis was corroborated with the results of Narasimhacharya et al. (2010), who suggested that the activity was directly related to the quantity of flavonoids, polyphenols, phenolic acids, protein and ascorbic acid content of the herb extract. ACE inhibitory activity of tulsi reported as 21% in aqueous extract by Nymen et al. (1998), while for ashwagandha it was in range of 11-22%, depending on type of extract used. The variation in the value of ashwagandha may be due to differences

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in the source of herb extract. Oboh et al. (2013) found that free phenolic compounds of garlic has slightly higher ACE inhibitory activity than bound phenolics, whereas Sendl et al. (1992) reported that the aqueous extract of the leaves of wild garlic contained glutamyl-peptides, which possess high ACE inhibitory activity.

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Results obtained were compared with the total polyphenols and total flavonoids that were estimated during the research. It was found that roselle had the maximum amount of

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total polyphenols followed by Emblica officinalis, ashwagandha, tulsi, ginkgo and garlic. Total flavonoids were the highest in Emblica officinalis followed by roselle, ashwagandha, tulsi, ginkgo and garlic (Table 6). Total polyphenols estimation could be used as an indicator

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for total antioxidant potential. But due to different configuration of active component results had shown variations among themselves. In addition our study demonstrated that Emblica officinalis showed fewer amounts of total polyphenols when compared to roselle however

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antioxidant activity obtained for Emblica officinalis was higher. This may be due to the important content of active component of Emblica officinalis that have high antioxidant

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power requiring less quantity to have a good antioxidant potential. Polyphenols of herbs are the complex mixture of compounds that may react with radicals by different mechanisms and

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often interact synergistically or by inhibition (Saucier and Waterhouse, 1999). The pathogenesis of a wide variety of clinical disorders is more prevalent due to free

radical oxidative stress. Potential antioxidant therapy includes natural free radical scavenging antioxidant agents (polyphenols) which are capable of augmenting the activity of enzymes such as superoxide dismutase, catalase and glutathione peroxidase. By virtue of its effect against free radicals, Emblica officinalis provides therapeutic intervention against oxidative threats in diseases. Ghosal et al. (1996) reported that tannins exhibit a very strong antioxidant activity in vitro and in vivo. Liu et al. (2008) observed that an antioxidant activity of Emblica 13

officinaliswas due to phenolic compounds such as geraniin, quercetin 3-b-D-glucopyranoside, kaempferol 3-b-D-glucopyranoside, isocorilagin, quercetin, and kaempferol. The main mechanism behind the cardio-protective action of Emblica officinalis is to inhibit the synthesis of HMG CoA (hepatic-3-hydroxy-3-methylglutaryl-Coenzyme A), a rate controlling enzyme in the metabolic pathway for the synthesis of cholesterol and other isoprenoids. The aqueous extract of Emblica officinalis fruit increased cardiac glycogen levels and decreased serum glutamic oxalo-acetic transaminase, glutamic pyruvic transaminase and LDL in rats having induced myocardial necrosis (Tariq et al., 1977). Emblica officinalis is a natural product that can be used as an effective antidyslipidaemic and

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antiatherogenic agent without any reported side effects (Antony et al., 2006). The results of the current study are proof of the potential of natural resources as inexpensive and safe approaches for hypertension/cardioprotection. 4. Conclusion

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The present study revealed that an aqueous extract of Indian gooseberry had the

highest antioxidant and ACE inhibitory potential when compared to ashwagandha, roselle,

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ginkgo, tulsi and garlic. A higher content of antioxidant molecules viz. phenolic compounds, flavonoids and vitamin C could possibly be attributed to the superior antioxidant potential of

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Emblica officinalis. The composition and type of phenolics and other antioxidants present in the six herbs tested may be the reason for differences in their reducing powers assessed using various methods. The results of the current study may be useful in formulating nutraceutical

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preparations and functional foods to address CVD related health problems. In a further study, the authors have encapsulated Emblica officinalis using a double emulsion technique, for its further application in functional food preparations (Chaudhary et al., 2019 & Chaudhary et

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al., 2020). Due to increased awareness among side effects of synthetic drugs, these plants

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have a potential for pharmaceutical applications.

Conflict of interest : NONE

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Acknowledgements The authors gratefully acknowledge the research grant (SERB/MoFPI/057/2015) provided by Science and Engineering Research Board, Ministry of Food Processing Industries, India. Sincere thanks to M/s. Plantae Herbal Extracts Pvt. Ltd., New Delhi, India for providing the herbal extracts. The first author thankfully acknowledge Director, NDRI for providing facilities and supporting in the form of Senior Research Fellowship of ICAR-

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NDRI, India.

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Solubility in water

Microbiological quality E. coli

Salmonell a spp.

ANHI1169

80.0%

Absent

Absent

ANAS1171

75.0%

Absent

Absent

Leaf

ANTU1175

90.0%

Absent

Absent

Fruit

ANAM1185

85.0%

Absent

Absent

Whole

KIL/CSHE75/GE 140507

-

Absent

Absent

Leaf

KIL/CSHE8/1105 99% 18

Absent

Absent

Anthocyanins (10%)

Flower

Withania somnifera (L.) Dunal

Withanolides (5.0%)

Root

Ocimum sanctum L.

Orintein (15%)

Emblica officinalis Gaertn.

Total Saponins By

Allium sativum L.

Allin (3.8%)

Ginkgo biloba L.

Ginkgo Flavone Glycosides (24.58%); Quercetin glycosides (12.36%); Kempferol glycosides (9.60%); Isorhamnetin glycosides (2.62%); Ginkgolic acid (6.64%)

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Hibiscus sabdriffa L.

Batch number

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Plant part used

pr

Active compound

e-

Herb

f

Table1. Detailed information of selected herbs used in the investigation

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Emblicanin (30%)

22

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Concentration of herbal extract

f

Table 2. Antioxidant activity of selected herbs determined using DPPH method Free radical scavenging activity (mmol/ml Trolox equivalent)

(µg/ml)

Emblica officinalis

Hibiscus sabdriffa

Withania somnifera

50

4.94±0.04aA

1.56±0.58bA

2.37±0.76cdA

100

5.84±0.40aB

2.68±0.81bB

200

8.24±0.64aC

2.47±0.29bB

250

9.42±0.20aD

500

Ocimum sanctum

1.85±0.06bdA

2.69±0.134cA

2.00±0.28bdAB

3.58±0.25cBC

2.01±0.06dAB

2.78±0.46bAB

1.96±0.19dAB

3.46±0.04cB

2.01±0.01bAB

3.85±0.44cB

2.10±0.34bB

2.69±0.73bB

3.26±0.01bcB

1.97±0.34cdAB

3.59±0.05eABC

1.61±0.37dA

11.26±0.20aE

2.78±0.17bB

3.96±0.03cC

2.09±0.15dAB

3.7±0.11eBC

1.89±0.15dAB

1000

11.39±0.15aE

4.20±0.21bC

4.91±0.10cD

1.58±0.63dA

3.62±0.23dABC

1.98±0.41dAB

5000

10.90±0.10aE

8.613±0.02bD

7.55±0.09cE

2.47±0.53deB

5.07±0.44eD

2.32±0.12dBC

9.81±0.40aD

9.89±0.07aE

10.28±0.23aF

2.45±0.19bB

6.43 ± 1.52cE

2.67±0.29bC

180.35±1.17

1362.22±1.18

4612.17±1.33

1034.87±1.25

4950.33±1.37

5068.66±1.53

e-

Pr

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IC50

pr

Ginkgo biloba

10000

Allium sativum

* Two way ANOVA by IBM SPSS Statistics 20 software Values are Mean ± SD (number of replicates =4) a, b, c, d, e - Means with different superscripts within a row differ significantly (p<0.05) A, B, C, D, E, F - Means with different superscripts within a column differ significantly (p<0.05) 22

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Ginkgo biloba

Ocimum sanctum

524.21±31.92dA

437.53±29.00eA

697.98±9.25fA

Allium sativum

pr

Antioxidant activity (µmol/ml Trolox equivalent) Concentration of herbal extract Withania Emblica officinalis Hibiscus sabdriffa (µg/ml) somnifera

f

Table 3. Antioxidant activity of selected herbs determined using ABTS method

1509.39± 17.91aA

1063.39±18.96bA

75

1688.51±55.31aB

1075.93±40.27bA

993.53±22.18cB

687.57±14.78dB

487.53±26.88eA

721.27±19.73dA

100

2166.76±9.25aC

1292.66±54.41bB

1237.13±38.08bC

774.25±30.98dC

822.59±38.69dB

712.31±17.06eA

200

3642.71±9.25aD

1453.87±29.83bC

1362.52±23.67cD

902.60±26.75dD

967.61±19.91eC

810.83±10.76fB

250

4221.26±14.77aE

1638.36±32.77bD

1527.31±36.71cE

1072.63±10.89dE

1084.29±11.38dD

796.50±37.17eB

500

4772.95±9.02aF

2625.31±37.89bE

2236.62±22.18cF

1209.31±8.61dF

1289.33±12.77eE

982.78±32.77fC

1000

4884.88±11.39aG

3726.37±38.11bF

3101.27±29.00cG

1321.00±14.78dG

1491.02±11.39eF

1120.97±45.67fD

5000

4989.90±28.03aH

3924.74±26.88bG

3316.31±53.76cH

1519.36±28.29dH

1631.05±26.88eG

1254.32±73.12fE

5123.25±17.64aI

4908.22±34.16bH

4026.42±60.38cI

1687.72±14.78dI

2539.52±52.62eH

2052.78±32.21fF

167.19±1.04

540.88±1.18

541.76±1.18

258.96±1.26

654.61±1.61

6649.22±1.31

IC50

Pr

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10000

918.30±12.24cA

e-

50

* Two way ANOVA by IBM SPSS Statistics 20 software Values are Mean ± SD (number of replicates=4) a, b, c, d, e, f - Means with different superscripts within a row differ significantly (p<0.05) A, B, C, D, E, F, G, H, I - Means with different superscripts within a column differ significantly (p<0.05)

22

Reducing activity (mmol/ml Ascorbic acid)

f

Emblica officinalis

Hibiscus sabdriffa

Withania somnifera

Allium sativum

Ginkgo biloba

Ocimum sanctum

50

1.78±0.08aA

0.31±0.04bA

pr

Concentration of herbal extract

oo

Table 4. Antioxidant activity of selected herbs determined using FRAP method

0.38±0.03bA

0.32±0.08bA

0.30±0.06bA

100

3.10±0.02aA

0.56±0.08bAB

0.53±0.09bA

0.46±0.08bA

0.44±0.16bcAB

0.32±0.04cA

200

7.09±0.07aA

0.90±0.13bBC

0.66±0.03bAB

0.41±0.05cA

0.47±0.10cAB

0.41±0.01cB

250

6.56±0.08aA

1.25±0.04bC

0.85±0.02cB

0.36±0.08dA

0.45±0.02eAB

0.35±0.01dA

500

13.57±0.52aB

3.02±0.04bD

0.95±0.06cB

0.33±0.03dA

0.56±0.03dB

0.42±0.04dB

1000

26.12±0.24aC

3.76±0.52bE

0.85±0.06cB

0.60±0.06dB

0.89±0.02cC

0.62±0.04dC

5000

122.57±6.54aD

14.20±0.28bC

7.27±0.24cC

1.31±0.13dC

2.01±0.18dD

2.10±0.05dD

30.01±0.87bG

13.61±0.40cD

2.41±0.12dD

3.61±0.04dE

2.89±0.04dE

10000

251.62±6.31aE

e-

0.39±0.03bA

Pr

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(µg/ml)

* Two way ANOVA by IBM SPSS Statistics 20 software Values are Mean ± SD (number of replicates =4) a, b, c, d, e - Means with different superscripts within a row differ significantly (p<0.05) A, B, C, D, E- Means with different superscripts within a column differ significantly (p<0.05)

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Inhibition (%)

10mg/ml

Emblica officinalis

82.08±5.18a

10mg/ml

Hibiscus sabdriffa

58.89±7.54bc

10mg/ml

Withania somnifera

62.35±3.70c

10mg/ml

Ocimum sanctum

22.87±6.24d

10mg/ml

Allium sativum

39.57±2.58bde

10 mg/ml

Ginkgo biloba

f

pr

Sample/ Herbal extract

Pr

e-

Concentration

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Table 5. Angiotensin converting enzyme inhibition activity of selected herbs

11.21±2.61df

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* One way ANOVA by IBM SPSS Statistics 20 software ** Captopril (a standard medicine) showed 31.64± 5.05% ACE enzyme inhibition at 0.01 µmol concentration Values are Mean ± SE (number of replicates=4) a, b, c, d, e, f - Means with different superscripts differ significantly (p<0.01)

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f Polyphenols (%)

Flavonoids (%)

Hibiscus sabdriffa

38.76±0.54

2.10±0.049

Withania somnifera

24.70±2.32

1.83±0.036

Ocimum sanctum

19.20±1.43

2.59±0.84

Emblica officinalis

37.60±2.11

5.60±0.41

Ginkgo biloba

1.56±0.62

Allium Sativum

0.72±0.03

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Pr

e-

pr

Herbs

oo

Table 6. Total polyphenols and flavonoids content of selected herbs

1.21±0.62 1.05±0.47

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Values are Mean ± SE (number of replicates=4)

22