Since prehistoric times, herbs were the basis for nearly all medicinal therapy until synthetic drugs were developed in the nineteenth century, a time when the prominence of herbal remedies gradually decreased in developed countries [
Free radicals are molecular species or atoms which contain one or more unpaired electrons. These are constantly produced in the human body as a result of cell metabolism [
Previous researches have indicated that phenolic compounds and flavonoids act as excellent anti-inflammatory agents [
A novel Polyherbal Ayurvedic Formulation (PHAF), which consists of powders of seven medicinal plants (Table
Composition of Polyherbal Ayurvedic Formulation (PHAF).
No. | Scientific name | Family | Amount and part of the plant used |
---|---|---|---|
1 | Fabaceae | Bark (10 g) | |
2 | Leguminosae | Stem (10 g) | |
3 | Menispermaceae | Stem (10 g) | |
4 | Cyperaceae | Rhizome (10 g) | |
5 | Zingiberaceae | Rhizome (10 g) | |
6 | Meliaceae | Bark (10 g) | |
7 | Bignoniaceae | Bark (10 g) |
Fresh plant materials required for PHAF were collected from Colombo (6° 55′ 54.98″ N
In brief, all the ingredients were washed thoroughly and air dried. Then ingredients were pulverized separately into a coarse powder and combined in a stainless steel vessel in a ratio of 1 : 1 (w/w) and mixed well.
PHAF powder (50 g) was added to a round bottom flask containing 150 mL of ethanol and boiled for 4 h. Then, the extract was filtered using Whatman 0.45
PHAF powder (50 g) was added to a round bottom flask containing 150 mL of water and boiled for 4 h. Then, the extract was filtered using Whatman 0.45
Physicochemical parameters of PHAF powder were assayed according to the World Health Organization (WHO) guidelines on quality control methods for herbal materials [
Phytochemical screening was carried out for PHAF (ethanol and aqueous extracts) according to methods described by Yadav and Agarwala [
Quantitative determination of arsenic (As) [
Microbial tests for aerobic plate count [
Antioxidant activity of the ethanol and aqueous extracts of PHAF powder was investigated using the following in vitro assays.
Total polyphenol content of ethanol and aqueous extracts of PHAF was determined using the Folin–Ciocalteu reagent [
Total flavonoid content of ethanol and aqueous extracts of PHAF was determined using the aluminium chloride method in 96-well microplates [
The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay was performed in 96-well microplates according to the method described by Blois [
The ABTS
The ORAC radical scavenging assay was performed in 96-well microplates according to the method described by Ou [
Data were statistically analyzed using GraphPad Prism version 4.0. One-way analysis of variance (ANOVA) and Duncan’s multiple range test were used to determine the differences among treatment means.
Physicochemical parameters of PHAF are illustrated in Table
Physicochemical parameters of Polyherbal Ayurvedic Formulation (PHAF).
Physicochemical parameters | Amount (% dry weight basis) |
---|---|
Moisture content | 5.6 ± 0.2 |
Total ash content | 6.5 ± 0.1 |
Water soluble ash content | 1.4 ± 0.1 |
Acid insoluble ash content | 0.9 ± 0.0 |
Hot-ethanol extractable matter | 10.5 ± 4.1 |
Cold-ethanol extractable matter | 8.4 ± 0.2 |
Hot-water extractable matter | 7.7 ± 0.2 |
Cold-water extractable matter | 3.9 ± 0.1 |
Data represented as mean ± SEM (standard error mean);
Tannins, flavonoids, saponins, coumarins, and phenolic compounds were present in both hot water and hot-ethanol extracts. Alkaloids and cardiac glycosides were present only in aqueous extract.
Heavy metals such as Pb, Cd, Hg, and As were not detected in PHAF. The minimum detection levels of Pb, Cd, Hg, and As were 0.5 ppm, 0.05 ppm, 0.05 ppm, and 0.05 ppm, respectively.
Microbes including
Antioxidant properties of ethanol and aqueous extracts of PHAF powder are given in Table
Antioxidant properties of Polyherbal Ayurvedic Formulation (PHAF).
Samples | Antioxidant properties | ||||
---|---|---|---|---|---|
TPC | TFC | ORAC | DPPH | ABTS | |
(mg gallic acid equivalents/g of extract) | (mg quercetin equivalents/g of extract) | (mg trolox equivalents/g of extract) | (mg trolox equivalents/g of extract) | (mg trolox equivalents/g of extract) | |
Ethanol extract | 327.07 ± 9.65 | 224.6 ± 8.42 | 1481.44 ± 30.20 | 227.17 ± 6.16 | 577.08 ± 5.48 |
Aqueous extract | 103.65 ± 4.94 | 76.6 ± 5.83 | 481.11 ± 17.30 | 79.50 ± 4.42 | 198.20 ± 4.55 |
Data represented as mean ± SEM (standard error mean).
In the present study, significantly more matter was extracted by ethanol than by water. Further, more extractable matter was present in hot extracts than in cold extracts (both water and ethanol). Very little acid insoluble ash was present in PHAF (Table
The main purpose of our study was to evaluate antioxidant activity of PHAF by using in vitro assays. Antioxidant activity has been evaluated previously for each of the seven plant species present in PHAF. Examples include antioxidant activities for
According to traditional Ayurveda practice, PHAF is administered to patients in the hot water extract form. We, therefore, assayed antioxidant activities for the aqueous extract. However, hot water preparations have some drawbacks, such as unpleasant taste, and have lower stability, requiring them to be prepared freshly on each occasion. To avoid this drawback, it would be better to develop pharmaceutics such as syrups and tonics. Therefore, we also assayed the ethanol extract of PHAF, which exhibited overall higher levels of antioxidant activities than that of aqueous extract (Table
In ABTS+ assay, a blue/green ABTS.+ chromophore is generated by the oxidation of ABTS with potassium persulfate. Antioxidants which have the ability of donating hydrogen reduced the blue/green color of ABTS+ and can be measured spectrophotometrically at 745 nm [
In the present study, IC50 values of ABTS+ (for both aqueous and ethanol extracts) were significantly lower than that of DPPH (Figure
IC50 values of aqueous and ethanol extracts of Polyherbal Ayurvedic Formulation (PHAF) for ABTS+ and DPPH assays.
The capacity of a compound to scavenge peroxyl radicals, generated by spontaneous decomposition of 2,2,-azobis(2-amidinopropane) dihydrochloride (AAPH), was estimated in the ORAC assay. The ORAC value is directly proportional to the degree of antioxidant power [
Phenolic compounds and flavonoids are reported to have antioxidant and free-radical scavenging activity [
The ash content is a criterion to judge the identity and purity of crude drugs [
The phytochemical constituents of many medicinal plants have been recorded by a number of researchers during the last few decades [
We have reported the quality assessment and antioxidant properties of PHAF for the first time. PHAF has potent antioxidant properties, which may be due to the presence of high content of polyphenols and flavonoids. Even though aqueous extract of PHAF is used in traditional practice, ethanol extract can be used to formulate pharmaceuticals such as tonics and syrups due to its potent antioxidant properties.
All the data obtained and materials analyzed in this research are available from the corresponding author upon request and also can be accessed from Ph.D. thesis of the corresponding author deposited at the University of Kelaniya, Sri Lanka.
The funding sponsor had no role in the study design, performance, data collection and analysis, decision to publish, or preparation/writing of the manuscript.
The authors declare that they have no conflicts of interest.
The authors would like to acknowledge Industrial Technology Institute for providing laboratory facilities for the research. This study was supported by the funding from University Grants Commission, Sri Lanka (UGC/ICD/CRF 2009/2/45).